Treatment of Inflammatory Bowel Disease with IFN-Gamma Inhibitors

ABSTRACT

The invention concerns a method for the prevention or treatment of inflammatory bowel disease by administering an interferon-γ inhibitor. The invention further concerns pharmaceutical compositions and bispecific molecules useful in such method.

BACKGROUND OF THE INVENTION

I. Field of the Invention

The invention concerns the prevention or treatment of inflammatory boweldisease by administering an interferon-gammon (IFN-γ) inhibitor.

II. Description of Background and Related Art

Inflammatory bowel disease (IBD) is a collective term for ulcerativecolitis (UC) and Crohn's disease, which are considered as two differententities, but have many common features and probably share at least somepathologic mechanisms. There is sufficient overlap in the diagnosticcriteria for UC and CD that it is sometimes impossible to say which agiven patient has; however, the type of lesion typically seen isdifferent, as is the localization. UC mostly appears in the colon,proximal to the rectum, and the characteristic lesion is a superficialulcer of the mucosa; CD can appear anywhere in the bowel, withoccasional involvement of stomach, esophagus and duodenum, and thelesions are usually described as extensive linear fissures.

The aetiology of these diseases is unknown and the initial lesion hasnot been clearly defined; however, patchy necrosis of the surfaceepithelium, focal accumulations of leukocytes adjacent to glandularcrypts, and an increased number of intraepithelial lymphocytes andcertain macrophage subsets have been described as putative earlychanges, especially in Crohn's disease.

The current therapy of IBD usually involves the administration ofantiinflammatory or immunosuppressive agents, such as sulfasalazine,corticosteroids, 6-mercaptopurine/azathioprine, or cyclosporine, whichusually bring only partial results. Ifanti-inflammatory/immunosuppressive therapies fail, colectomies are thelast line of defense. About 30% of CD patients will need surgery withinthe first year after diagnosis. In the subsequent years, the rate isabout 5% per year. Unfortunately, CD is characterized by a high rate ofoccurrence; about 5% of patients need a second surgery each year afterinitial surgery. In UC, a further reason for resorting to surgery isthat the patients are known to be at much increased risk for developingcolorectal cancer, starting 10-15 years after the diagnosis ofulcerative colitis. Presumably this is due to the recurrent cycles ofinjury to the epithelium, followed by regrowth, increasing the risk oftransformation. Accordingly, colostomy is used as prophylaxis againstthe development of cancer in UC patients.

IBD is rather common, with a prevalence that is claimed to be in therange of 70-170 in a population of 100,000. In view of the apparentshortcomings of the present treatments, there is a great medical needfor a non-surgical approach, based upon a better understanding of theimmunological reasons underlying this disorder.

A recent review of the characteristics of the mucosal immune system inIBO is by Brandtzaeg, P. et al. on pages 19-40 in Immunology ofGastrointestinal Disease, MacDonald, T. T. ed., Immunology and MedicineSeries, Volume 19, Kluwer Academic Publishers, 1992.

Several attempts have been made to identify factors instrumental in theinitiation of IBD; there have been reports on a genetically determinedmucin defect in UC, and increased intestinal permeability and/ordefective mucosal IgA system in Crohn's disease. There have further beenpersistent attempts to identify infectious agents associated with eitherUC or CD, with not much success. According to a recent, rathercontroversial theory, CD may be a vascular disease, characterized bylocalized tendency to thrombus formation leading to multifocalintestinal infarction and thereby causing the occurrence of earlylesions. Altogether, the nature of the initial insult(s) resulting inIBD remains to be identified. However, there is considerable evidencethat hyperactivation of the mucosal immune system in the gut throughvarious immunopathological mechanisms may cause established IBD lesions.

The gut immune system is special in that the gut must absorb a vastamount of potentially antigenic material (food proteins) withoutreacting to any of it, and must control reactions to non-pathogenicorganisms such as normal gut flora without losing the ability to reactto abnormal, replicating organisms. The regulatory mechanisms that allowthis kind of selective response are almost completely unknown. It isalso unclear whether IBD results from an appropriate immune response toan abnormally persistent antigen, or an inappropriate response to anormal antigen.

The major lymphocytic tissues in the small intestine are the so calledPeyer's patches (PP). Unlike the lymph node, PP do not have a capsule ofafferent lymphatics. The epithelium over the PP lacks the crypts andvilli of normal gut epithelium and is referred to as follicle-associatedepithelium (FAE) containing cells called M cells. These are the majorroute of antigen transfer into the PP, and allow for direct sampling ofantigen from the gut lumen by pinocytosis. Antigen is transported fromthe epithelium and presented to immunocompetent B cells, macrophages anddendritic cells in the underlying area. The colon has similar lymphoidarrangements called the lymphoid follicles. Lymphoid follicles are notidentical to PP, but also have specialized epithelium containing Mcells, and probably function as antigen presenting sites.

Underneath the epithelium there is a tissue called the lamina propriawhich forms the core of the villus and is densely infiltrated withlymphocytes bearing homing receptors which selectively bind to themucosal lymphoid high endothelium. B cells comprise about 50% of thelymphocytes in the lamina propria of the gut, whereas the other half oflymphocytes are CD3+T cells most of which are also CD4+. In the normalintestine, most of the B cells in the lamina propria are IgA+, althoughIgM-, IgG- and IgD-expressing cells are also found. Most of theimmunoglobulin secreted into the intestine is IgA, and half of that isIgA-2, in contrast to the lymph nodes where most of the secreted IgA isof the IgA-1 isotype. The abundance of IgA antibodies is probablycrucial for immunological homeostasis within the lamina propria. IgAantibodies lack potent effector functions such as complement activation,and may therefore block non-specific biological amplification mechanismstriggered by locally produced or serum-derived IgG antibodies.

As already mentioned, CD3+T cells comprise approximately half of thelymphocytes in the lamina propria. This phenotype is also prevalent inhuman PP, and specifically in the interfollicular zones surrounding thehigh endothelial venules (HEV). In contrast, CD8+T lymphocytes arepredominant in the epithelium of humans.

Although it is not clear how inductive and suppressive immunoregulatorymechanisms are achieved in the gut, the lamina propria and epithelium,along with the organized lymphoepithelial nodules and the largerlymphoid aggregates, e.g. PP, are probably all involved in a complexmanner.

The established mucosal IBD lesions are dominated byimmunoglobulin-producing cells, both in UC and in CD. However, while theIgA- and IgM-expressing cell populations only increase several times ascompared to normal mucosa, there is a disproportionate rise in thenumber of IgG-producing immunocytes. The actual number depends on theseverity of the disease, but both UC and CD are characterized by adramatically increased IgG production, including selective increases inthe levels of specific IgG isotypes in both the intestinal mucosa andperipheral blood, and consequently by a remarkable decrease in theIgA/IgG ratio (MacDermott, R. P. et al., Gastroenterology 96, 764-768(1989)].

It has been tentatively suggested that the selective increase in theproduction of IgG might reflect an immune response to one or severalantigens, as well as the balance of cytokines and other regulatoryfactors, such as transforming growth factor β (TGF-β), that modulateimmunoglobulin production in different populations of B cells (PodoIsky,D. K., New England J. Med. 325, 928-937 (1992)]; however, there is nosatisfactory explanation for this phenomenon as of yet.

Changes in the major subsets of the T cells population have also beenobserved in IBD. In CD, there is evidence that there are more memorycells than normal (lacking the CD45RA marker) and increased EL-2Rexpressing (activated) cells. CD4+T-cells in the lamina propria seem tobe increased relative to CD8+T-cells in UC. It has been observed thatthe expression of certain cytokines is increased in IBD. This includesincreased expression of interleukin-1 (IL-1), interleukin-6 (IL-6),altered expression of interleukin-2 (IL-2) and its receptor in bothtissue and the circulation (Mahida, Y. R. et al., Gut 30, 838-838(1989); Kusugami, K. et al., Gastroenterology 97, 1-9 (1989); Ugumsky,M. et al., Gut 31, 686-689 (1990)]. Curiously, lower levels of IL-2 andIL-2 receptors have been reported in tissue from certain patientsdiagnosed with CD (Kusugami et al., supra). An IL-1 receptor antagonisthas been described to reduce the severity of inflammation in a rabbitmodel of colitis [Cominelli, F. et al., J. Clin. Invest. 86, 972-980(1990)]. A remarkably intensified epithelial expression of humanleukocyte antigen complex DR (HLA-DR) in both UC and CD has led to theproposal that various cytokines are released locally from activated Tcells (Selby, W. S. et al., Clin. Exp. Immunol. 53, 614-618 (1983);Brandtzaeg, P. et. al. Ann. Gastroenterol. Hepatol. 21, 201-220 (1985);Rognum, T. O. et. al., Gut 23, 123-133 (1982); Fais, S. et al., Clin.Exp. Immunol. 605-612 (1987)]. Although both IFN-γ and tumor necrosisfactor α (TNF-α) are capable of enhancing epithelial HLA-DR onintestinal epithelium, difficulties have been encountered indemonstrating the production of IFN-γ in the IBD lesion [MacDonald, T.T. et al., Clin. Exp. Immunol. 81, 301-305 (1990)]. On the other hand, araised number of cells producing TNF-α has been observed for both UC andCD lesions (MacDonald et al., supra).

In conclusion, whereas the relatively reduced IgA production and thestriking increase of IgG-producing cells in IBD may reflect theestablishment of a local immune defense mechanism, the causative factorsfor these changes have not yet been determined. Similarly, despitesuggestions that certain cytokines and cytokine receptors may play arole in the development of IBD, their individual roles and complexinteractions are not well understood.

It is an object of the invention to provide a method for the prophylaxisor treatment of inflammatory bowel disease (IBD), including ulcerativecolitis (UC) and Crohn's disease (CD).

It is a further object to provide bispecific molecules comprising anIFN-γ inhibitor and a further specificity to a target involved in theinitiation or development of IBD.

It is yet another object to provide a method for the use of IFN-γinhibitors in the preparation of pharmaceutical compositions suitablefor the prophylaxis or treatment of disorders that involve a reductionin the percentage of IgA-producing immunocytes, such as IBD.

These and further objects of the present invention will be apparent forone skilled in the art.

SUMMARY OF THE INVENTION

The present invention is based on the premise that increased productionof IFN-γ is instrumental in the inflammation, increased expression ofHLA-DR on epithelia, and the change of IgA:IgG ratios in the gut in IBDpatients. IFN-γ probably reduces the relative amount of immunoglobulinof the IgA subtype by selective killing of IgA-producing B cells, inparticular CD5+B cells, which constitute about 50% of the IgA-expressingB cells in the gut; it is, however, not intended to be bound by this orby any other theory.

In one aspect, the invention concerns a method comprising administeringto a patient having or at risk of developing an inflammatory boweldisease, such as ulcerative colitis or Crohn's disease, atherapeutically or preventatively effective amount of an IFN-γinhibitor. The IFN-γ inhibitor may, for example, be an amino acidsequence from an anti-IFN-γ antibody, an IFN-γ receptor polypeptide, ananti-IFN-γ receptor antibody, or an IFN-γ variant. The method includesthe treatment of humans and non-human animals, such as mammals, rodents,etc.

In a particular embodiment, the IFN-γ inhibitor comprises theextracellular domain of an IFN-γ receptor, optionally fused to a stableplasma protein. The stable plasma protein preferably is animmunoglobulin, and the fusion preferably comprises at least a hingeregion and the CH2 and CH3 domains of an immunoglobulin heavy chain.

In another aspect, the invention concerns a bispecific moleculecomprising an IFN-γ inhibitor amino acid sequence and a further aminoacid sequence capable of binding a target involved in the initiation ordevelopment of IBD. Just as before, the IFN-γ inhibitor may be an IFN-γreceptor, an anti-IFN-γ antibody, an anti-IFN-γ receptor antibody and anIFN-γ variant, and the further amino acid sequence preferably is from anIFN-γ inhibitor different from the one providing the first specificity,an IL-1 inhibitor, a TNF-α inhibitor, a CD11a/18 inhibitor, a CD11b/18(VLA4) inhibitor, or an L-selectin inhibitor.

In a specific embodiment, the bispecific molecule is a bispecificimmunoadhesin.

In a further aspect, the invention concerns nucleotide sequencesencoding the bispecific molecules of the invention, expression vectorscontaining such nucleotide sequences, recombinant host cells transformedwith the expression vectors, and processes for culturing such host cellsso as to express the encoded bispecific molecules.

In a still further aspect, the invention concerns the use of IFN-γinhibitors in the preparation of pharmaceutical compositions for theprevention or treatment of disorders involving a reduction in thepercentage of IgA-producing lymphocytes.

Production of IFN-γ at inappropriate levels, locations, or developmentalstages has been implicated in the pathogenesis of several autoimmune andinflammatory diseases and in graft rejection. Thus, IFN-γ was present innewly diagnosed diabetic children and in muscle biopsies from patientswith polymyosis. It has also found to cause exacerbation of autoimmunediseases such as multiple sclerosis and psoriasis. Anti-IFN-γ antibodieswere shown to delay the development of a lupus-like disease with fatalimmune-complex glomerulonephritis in mice, to inhibit endotoxin-inducedlethality, and, alone or in combination with immunosuppressive agents,to inhibit allograft rejection. Fusion proteins consisting of the mouseIFN-γ receptor extracellular portion and constant domains ofimmunoglobulin molecules have been made and proposed as useful in thetherapy for autoimmune diseases, chronic inflammation, delayed type ofhypersensitivity and allograft rejection [Kurschner, C. et al., J. Biol.Chem. 267, 9354-9360 (1992); Dembic, Z et al., The 1992 ISIR Meeting ofthe Interferon System, Toronto, Ontario, Canada, Sep. 28-Oct. 2, 1992,J. of Interferon Research Vol. 12, suppl. 1 Sep. 1992, Abstract P7-3].Nowhere has been proposed, however, that IFN-γ may play a major role inthe development of IBD, and, as mentioned before, attempts todemonstrate the production of IFN-γ in IBD lesions were unsuccessful.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1(A) Schematic structure of IFNγR-IgG immunoadhesins. IFNγR (shadedbar) denotes the extracellular portion of the human or murine IFN-γreceptor and IgG-1 (thin line) denotes the hinge region and CH2 and CH3constant domains of human IgG-1 heavy chain. Locations of putativeN-linked glycosylation sites are shown (square boxes). (B-D) Subunitstructure of IFNγ-IgG. HEK293 cells were transfected with a vectordirecting transient expression of murine (lanes 1, 2) or human (lanes 3,3) IFNγR-IgG. The protein was recovered from culture supernatants andpurified by affinity chromatography on S. aureus protein A. SDSpolyacrylamide electrophoresis was carried out without (−) or with (+)reduction by 10 mM dithiothreitol (DTT). The proteins were stained withCoomassie blue (B) or electroblotted onto introcellulose paper andincubated with (C) antibodies to human IgG Fc or with (D) murine (lanes1, 2) or human (lanes 3, 4) [¹²⁵I]IFN-γ (10 nM) alone (lanes 1, 3), orwith 1 μM unlabeled human or murine IFNγ (lanes 2, 4). Blots weredeveloped with horseradish peroxidase-conjugates second antibody and4-chloronaphtol (C) or by autoradiography (D).

FIG. 2 Binding of IFNγR-IgG to IFNγ. Human (A) or murine (B) IFNγR-IgGwas immobilized in microtiter wells coated with anti-IgG Fc antibody andincubated with increasing concentrations of recombinant human or murine[¹²⁵I]IFN-γ, respectively. Scatchard analyses of the saturation data areshown in insets. Nonspecific binding was determined by omitting theIFNγR-IgG and was typically less than 10% of the total binding. The dataare from a representative experiment done in triplicate.

FIG. 3 Inhibition of IFN-γ by IFNγR-IgG in vitro. (A) Inhibition ofhIFN-γ induction of ICAM-1 expression in human HeLa cells by hIFNγR-IgG(closed circles). (B) Inhibition of mIFN-γ induction of the class I MHCantigen H2-K^(k) in mouse L929 cells by mIFNγR-IgG (open circles). (C)Inhibition of hIFN-γ antiviral activity by hIFNγR-IgG as measured bysurvival or human A 549 cells infected by encephalomyocarditis virus(EMCV). (D) Inhibition of mIFN-γ antiviral activity by mIFNγR-IgG asmeasured by survival of mouse L929 cells infected by EMCV. The data ineach panel are means±SD from two experiments in which the number ofreplicates was 1 (A,B) or 4 (C,D). In each respective panel, CD4-IgG(open boxes) (A,B), or mIFNγR-IgG (C), or hIFNγR-IgG (d) were used asnegative controls.

FIG. 4 Inhibition of IFN-γ by IFNγR-IgG in a mouse model forListeriosis. Mice were injected with vehicle (open bars), 200 μg ofCD4-IgG (shaded bars), or 20 μg of mIFNγR-IgG (closed bars) followedimmediately by 4×10⁴ viable Listeria monocytogenes. Three days later,spleen and liver homogenates were subcultured for 24 hours andcolony-forming units (CFU) were enumerated. Data are means±SEM (n=4 miceper group) from a representative experiment.

FIG. 5 shows the amount of glycosaminoglycans (GAGs) in frozen sectionsof human fetal small intestines, as visualized by silver staining.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

“Inflammatory bowel disease (IBD)” is used as a collective term for“ulcerative colitis (UC) and “Crohn's disease (CD)”. Although UC and CDare generally considered as two different entities, their commoncharacteristics, such as patchy necrosis of the surface epithelium,focal accumulations of leukocytes adjacent to glandular crypts, and anincreased number of intraepithelial lymphocytes (IEL) and certainmacrophage subsets, justify their treatment as a single disease group.

The term “reduction in the percentage of immunoglobulin A (IgA)” is usedto refer to a decrease in the relative amount of antibodies of the IgAclass in the patient's body, as compared to antibodies of other classes,i.e. IgG, IgM, IgD, IgE, in particular IgG, whether due to a reductionin the percentage of IgA-producing immunocytes or to their decreasedability to produce IgA, or to any other factor(s). If the condition tobe treated is IBD, the reduction takes place within the gut.

Interferon gamma (IFN-γ), also known as immune interferon, is a memberof the interferon family, which exhibits the antiviral andanti-proliferative properties characteristic of interferons-α and -βbut, in contrast to those interferons, is pH 2 labile. IFN-γ wasoriginally produced upon mitogenic induction of lymphocytes. Therecombinant production of human IFN-γ was first reported by Gray,Goeddel and co-workers [Gray et al., Nature 295, 503-508 (1982)], and issubject of U.S. Pat. Nos. 4,762,791, 4,929,544, 4,727,138 and 4,925,793.The recombinant human IFN-γ of Gray and Goeddel as produced in E. coli,consisted of 146 amino acids, the N-terminal portion of the moleculecommencing with the sequence CysTyrCys. It has later been found that thenative human IFN-γ (i.e., that arising from mitogen induction of humanperipheral blood lymphocytes and subsequent purification) is apolypeptide which lacks the CysTyrCys N-terminus assigned by Gray etal., Supra. As used herein, “interferon gamma” or “IFN-γ” refersvariously to all forms of (human and non-human animal) interferon gammaas are known to be biologically active in accepted interferon gammaassays, such as by inhibibon of virus replication in a suitable cellline (inhibibon of encephalomyocarditis virus replication in human lungcarcinoma cell line A549 for human IFN-γ), induction of class IIantigens, heat liability, other antiviral, antitumor or immunoregulatoryassays, or neutralization by antibodies having immunoreactivity forgamma interferon but not alpha- or beta-interferon, and is meant toinclude human interferon-γ in a mature, pro, met or des(1-3) (alsoreferred to as desCysTyrCys IFN-γ) form, whether obtained from naturalsource, chemically synthesized or produced by techniques of recombinantDNA technology. A complete description of the preparation of recombinanthuman interferon gamma (h IFN-γ) including its cDNA and amino acidsequences is shown in the United States Patents cited hereinabove (e.g.U.S. Pat. No. 4,762,791). CysTyrCys-lacking recombinant human IFN-γ,including variously truncated derivatives are, for example, disclosed inEuropean Publication No. 146,354. Non-human animal interferons,including animal, such as mammalian IFN-γ, are, for example, disclosedin European Publication No. 88,622. The term includes variouslyglycosylated forms and other variants and derivatives of suchinterferons, whether known in the art or will become available in thefuture. Examples of such variants are alleles, and the products of sitedirected mutagenesis in which residues are deleted, inserted and/orsubstituted (see, for example European Publication No. 146,354 referredto above).

The expressions “human interferon gamma”, “human IFN-γ” and “hIFN-γ”,which are used interchangeably, refer to a family of polypeptidemolecules that comprise the full-length (146 amino acids) human IFN-γ ofGray et al., supra, the native human IFN-γ lacking the first threeN-terminal amino acids of the full length species (desCysTyrCys humanIFN-γ), and their amino acid sequence variants, provided that thenucleotide sequences encoding such variants are capable of hybridizingunder stringent conditions with the complement of a nucleotide sequenceencoding the native amino acid sequence, and that they retain theability to exhibit IFN-γ biological action. This definition specificallyincludes human IFN-γ from natural sources, synthetically produced invitro or obtained by genetic manipulation including methods ofrecombinant DNA technology. The amino acid sequence variants preferablyshare at least about 65% sequence homology, more preferably at leastabout 75% sequence homology, even more preferably at least about 85%sequence homology, most preferably at least about 90% sequence homologywith any domain, and preferably with the receptor binding domain(s), ofthe native human IFN-γ amino acid sequence. The definition specificallycovers variously glycosylated and unglycosylated forms of native humanIFN-γ and of its amino acid sequence variants.

The expressions “native human interferon gamma”, “native human IFN-γ”and “native hIFN-γ” are used to refer to the mature 143 amino acidsnative human IFN-γ that arises from mitogen induction of humanperipheral blood lymphocytes and subsequent purification, and anynaturally occurring fragment or derivative thereof provided that itexhibits IFN-γ biological action. This definition specifically includesnaturally occurring alleles of IFN-γ.

The expressions “interferon gamma inhibitor” and “IFN-γ inhibitor” areused interchangeably and refer to polypeptides or organic moleculescapable of inhibiting the interaction of native IFN-γ with its receptorand thereby blocking the pathogenic effect of native IFN-γ in at leastan in vitro model of inflammatory bowel disease, irrespective of themechanism by which this inhibition is achieved. IFN-γ inhibitorsspecifically include molecules capable of inhibiting the binding ofnative IFN-γ to its native receptor, either by binding to native IFN-γor by binding to a native IFN-γ receptor (IFN-γ antagonists), in astandard competitive binding assay. IFN-γ inhibitors blocking IFN-γaction by binding native IFN-γ include, but are not limited to, IFN-γreceptors, and (blocking) anti-IFN-γ antibodies. Alternatively, IFN-γantagonists may act by binding but not activating a native IFN-γreceptor, such as certain IFN-γ derivatives and anti-IFN-γ receptorantibodies. The term “inhibitor” is used in an analogous manner inrelation to other molecules, such as cytokines, e.g. IL-1, or TNF-α;CD11a/18; CD1b/18 (VLA-4); L-selectin.

IFN-γ receptors have been purified from different human (Aguet, M. &Merlin, G., J. Exp. Med. 165, 988-999 (1987); Novick, D. et al., J.Biol. Chem. 262, 8483-8487 (1987); Calderon, J. et al., Proc. Natl.Acad. Sci USA 85, 4837-4841 (1988)] and murine [Basu, M. et al., Proc.Natl. Acad. Sci. USA 85, 6282-6286 (1988)] cell types, and have beencharacterized as 90- to 95-kDa single chain integral membraneglycoproteins that display certain structural heterogeneity due to cellspecific glycosylation. The primary sequence of human IFN-γ receptor hasbeen elucidated by Aguet et al., Cell 55, 273-280 (1988), who cloned,expressed and sequenced a 2.1 kb human IFN-γ receptor cDNA from a Rajucell expression library prepared in λgt1 1. The cloning and expressionof the cDNA for the murine interferon gamma (IFN-γ) receptor wasreported by Gray, P. W. et al., Proc. Natl. Acad. Sci. USA 86, 8497-8501(1989). The terms “interferon gamma receptor” and ” IFN-γ receptor” areused interchangeably and refer to a family of polypeptide molecules thatcomprise any naturally occurring (native) IFN-γ receptor from any animalspecies, and amino acid sequence and glycosylation variants of suchreceptors, provided that the nucleotide sequences encoding such variantsare capable of hybridizing, under stringent conditions, to thecomplement of a nucleotide sequence encoding a native IFN-γ receptor,and that they retain the ability to bind IFN-γ. The amino acid sequencevariants preferably share at least about 65% sequence homology, morepreferably at least about 75% sequence homology, even more preferably atleast about 85% sequence homology, most preferably at least about 90%sequence homology with any domain, and preferably with the ligandbinding domain(s), of a native IFN-γ receptor amino acid sequence fromthe same (human or non-human) animal species. IFN-γ receptors(occasionally referred to as IFN-γ binding proteins) are disclosed, e.g.in EP 240,975 published 14 Oct. 1987; EP 369,413 published 23 May 1990;EP 393,502 published 24 Oct. 1990; EP 416,652 published 13 Mar. 1991.

The expressions “human interferon gamma receptor” and “human IFN-γreceptor”, which are used interchangeably, refer to a family ofpolypeptide molecules that comprise the full-length, native human IFN-γreceptor having the amino acid sequence reported by Aguet et al., supra,and its amino acid sequence variants, provided that the nucleic acidsencoding such variants are capable of hybridizing under stringentconditions with the complement of a nucleic acid encoding the nativeamino acid sequence, and that they retain the ability to bind IFN-γ.This definition specifically encompasses soluble forms of nativefull-length human IFN-γ receptor, from natural sources, syntheticallyproduced in vitro or obtained by genetic manipulation including methodsof recombinant DNA technology. The amino acid sequence variantspreferably share at least about 65% sequence homology, more preferablyat least about 75% sequence homology, even more preferably at leastabout 85% sequence homology, most preferably at least about 90% sequencehomology with any domain, and preferably with the ligand (IFN-γ) bindingdomain(s), of the native full-length human IFN-γ receptor amino acidsequence. The definition specifically covers variously glycosylated andunglycosylated forms of -any native human IFN-γ receptor and of itsamino acid sequence variants.

The “stringent conditions” are overnight incubation at 42° C. in asolution comprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodiumcitrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10%dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA.

The term “native human interferon gamma receptor” or “native human IFN-γreceptor” is used to refer to the mature full-length native human IFN-γreceptor as disclosed by Aguet et al., supra, and any naturallyoccurring fragment or derivative, such as allelic variant, thereofprovided that it retains the ability to bind IFN-γ.

“Biologically active” IFN-γ variants, also referred to as variantsexhibiting “IFN-γ biological activity” share an effector function of anaturally occurring IFN-γ molecule, which may, but need not, in additionpossess an antigenic function. Effector functions include receptorbinding, any anti-viral, anti-bacterial, cytotoxic, anti-tumor, orimmunoregulatory activity of a native IFN-γ molecule. The antigenicfunctions essentially mean the possession of an epitope or antigenicsite that is capable of cross-reacting with antibodies raised against anaturally occurring IFN-γ molecule. Molecules inhibiting “IFN-γbiological activity” inhibit an effector function of IFN-γ.

The terms “amino acid” and “amino acids” refer to all naturallyoccurring L-α-amino acids. The amino acids are identified by either thesingle-letter or three-letter designations: Asp D aspartic acid Thr Tthreonine Ser S serine Glu E glutamic acid Pro P proline Gly G glycineAla A alanine Cys C cysteine Val V valine Met M methionine Ile Iisoleucine Leu L leucine Tyr Y tyrosine Phe F phenylalanine His Hhistidine Lys K lysine Arg R arginine Trp W tryptophan Gln Q glutamineAsn N asparagine

These amino acids may be classified according to the chemicalcomposition and properties of their side chains. They are broadlyclassified into two groups, charged and uncharged. Each of these groupsis divided into subgroups to classify the amino acids more accurately:

I. Charged Amino Acids

Acidic Residues: aspartic acid, glutamic acid

Basic Residues: lysine, arginine, histidine

II. Uncharged Amino Acids

Hydrophilic Residues: serine, threonine, asparagine, glutamine

Aliphatic Residues: glycine, alanine, valine, leucine, isoleucine

Non-polar Residues: cysteine, methionine, proline

Aromatic Residues: phenylalanine, tyrosine, tryptophan

The term “amino acid sequence variant” refers to molecules with somedifferences in their amino acid sequences as compared to a native aminoacid sequence.

“Homology” is defined as the percentage of residues in the candidateamino acid sequence that are identical with the residues in the aminoacid sequence of their native counterparts after aligning the sequencesand introducing gaps, if necessary, to achieve the maximum percenthomology. Methods and computer programs for the alignment are well knownin the art.

Substitutional variants are those that have at least one amino acidresidue in a native sequence removed and a different amino acid insertedin its place at the same position. The substitutions may be single,where only one amino acid in the molecule has been substituted, or theymay be multiple, where two or more amino acids have been substituted inthe same molecule.

Insertional variants are those with one or more amino acids insertedimmediately adjacent to an amino acid at a particular position in anative sequence. Immediately adjacent to an amino acid means connectedto either the α-carboxy or α-amino functional group of the amino acid.

Deletional variants are those with one or more amino acids in the nativeamino acid sequence removed. Ordinarily, deletional variants will haveone or two amino acids deleted in a particular region of the molecule.

The term “glycosylation variant” is used to refer to a glycoproteinhaving a glycosylation profile different from that of a nativecounterpart. Glycosylation of polypeptides is typically either N-linkedor O-linked. N-linked refers to the attachment of the carbohydratemoiety to the side-chain of an asparagine residue. The tripeptidesequences, asparagine-X-serine and asparagine-X-threonine, wherein X isany amino acid except proline, are recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.O-linked glycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be involved in O-linked glycosylation. Anydifference in the location and/or nature of the carbohydrate moietiespresent in an IFN-γ receptor protein as compared to its nativecounterpart is within the scope herein.

“Stable plasma proteins” are proteins typically having about 30 to about2000 residues, which exhibit in their native environment an extendedhalf-life in the circulation, i.e. a half-life greater than about 20hours. Examples of suitable stable plasma proteins are immunoglobulins,albumins (such as human serum albumin, HSA), lipoproteins,apolipoproteins and transferrin.

Antibodies (Abs) and immunoglobulins (Igs) are glycoproteins having thesame structural characteristics. While antibodies exhibit bindingspecificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules which lack antigenspecificity. Polypeptides of the latter kind are, for example, producedat low levels by the lymph system and at increased levels by myelomas.

Native antibodies and immunoglobulins are usually heterotetramericglycoproteins of about 150,000 daltons, composed of two identical light(L) chains and two identical heavy (H) chains. Each light chain islinked to a heavy chain by one covalent disulfide bond, while the numberof disulfide linkages varies between the heavy chains of differentimmunoglobulin isotypes. Each heavy and light chain also has regularlyspaced intrachain disulfide bridges. Each heavy chain has at one end avariable domain (V_(H)) followed by a number of constant domains. Eachlight chain has a variable domain at one and (V_(L)) and a constantdomain at its other end; the constant domain of the light chain isaligned with the first constant domain of the heavy chain, and the lightchain variable domain is aligned with the variable domain of the heavychain. Particular amino acid residues are believed to form an interfacebetween the light and heavy chain variable domains [Clothia et al., J.Mol. Biol. 186, 651-663 (1985); Novotny and Haber, Proc. Natl. Acad.Sci. USA 82, 4592-4596 (1985)].

The variability is not evenly distributed through the variable regionsof antibodies. It is concentrated in three segments calledcomplementarity determining regions (CDRs) or hypervariable regions bothin the light chain and the heavy chain variable regions. The more highlyconserved portions of variable domains are called the framework (FR).The variable domains of native heavy and light chains each comprise fourFR regions, largely adopting a β-sheet configuration, connected by threeCDRs, which form loops connecting, and in some cases forming part of,the β-sheet structure. The CDRs in each chain are held together in closeproximity by the FR regions and, with the CDRs from the other chain,contribute to the formation of the antigen binding site of antibodies[see Kabat, E. A. et al., Sequences of Proteins of ImmunologicalInterest National Institute of Health, Bethesda, Md. (1987)]. Theconstant domains are not involved directly in binding an antibody to anantigen, but exhibit various effector functions, such as participationof the antibody in antibody-dependent cellular toxicity.

Papain digestion of antibodies produces two identical antigen bindingfragments, called Fab fragments, each with a single antigen bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a complete antigenrecognition and binding site. This region consists of a dimer of oneheavy and one light chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen binding site on thesurface of the V_(H)-V_(L) dimer. Collectively, the six CDRs conferantigen binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (C_(H)1) of the heavy chain. Fab′fragments differ from Fab fragments by the addition of a few residues atthe carboxy terminus of the heavy chain C_(H)1 domain including one ormore cysteines from the antibody hinge region. Fab′-SH is thedesignation herein for Fab′ in which the cysteine residue(s) of theconstant domains bear a free thiol group. F(ab′)₂ antibody fragmentsoriginally were produced as pairs of Fab′ fragments which have hingecysteines between them. Other, chemical couplings of antibody fragmentsare also known.

The light chains of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa and lambda (λ), based on the amino acid sequences of theirconstant domains.

Depending on the amino acid sequence of the constant region of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG andIgM, and several of these may be further divided into subclasses(isotypes), e.g. IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. Theheavy chain constant regions that correspond to the different classes ofimmunoglobulins are called α, delta, epsilon, γ, and μ, respectively.The subunit structures and three-dimensional configurations of differentclasses of immunoglobulins are well known. IgA-1 and IgA-2 are monomericsubclasses of IgA, which usually is in the form of dimers or largerpolymers. Immunocytes in the gut produce mainly polymeric IgA (alsoreferred to poly-IgA including dimers and higher polymers). Suchpoly-IgA contains a disulfide-linked polypeptide called the “joining” or“J” chain, and can be transported through the glandular epitheliumtogether with the J-chain-containing polymeric IgM (poly-IgM),comprising five subunits.

The term “antibody” is used herein in the broadest sense andspecifically covers single monoclonal antibodies, immunoglobulin chainsor fragments thereof, which react immunologically with a correspondingpolypeptide, such as IFN-γ or an IFN-γ receptor as well as anti-IFN-γand anti-IFN-γ receptor antibody compositions with polyepitopicspecificity, which have such properties.

The term “monoclonal antibody” as used herein refers to an antibody (ashereinabove defined) obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally occurringmutations that may be present in minor amounts. Monoclonal antibodiesare highly specific, being directed against a single antigenic site.Furthermore, in contrast to conventional (polyclonal)-antibodypreparations which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen. In addition totheir specificity, the monoclonal antibodies are advantageous in thatthey are synthesized by the hybridoma culture, uncontaminated by otherimmunoglobulins.

“Humanized” forms of non-human (e.g. murine) antibodies areimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin.For the most part, humanized antibodies are human immunoglobulins(recipient antibody) in which residues from a complementary determiningregion (CDR) of the recipient are replaced by residues from a CDR of anon-human species (donor antibody) such as mouse, rat or rabbit havingthe desired specificity, affinity and capacity. In some instances, Fvframework residues of the human immunoglobulin are replaced bycorresponding non-human residues. Furthermore, humanized antibody maycomprise residues which are found neither in the recipient antibody norin the imported CDR or framework sequences. These modifications are madeto further refine and optimize antibody performance.

The phrase “bispecific molecule” is used to define a polypeptide withtwo specificities. The amino acid sequences providing the twospecificities (“binding domains”) may be directly fused to each other ormay be connected with a “linker”. The linker may be the residue of acovalent cross-linking agent capable of linking the two binding domainswithout the impairment of their ability to bind their respective nativereceptors or ligands or a linkage the formation of which is induced bysuch cross-linking agents. A concise review of covalent cross-linkingreagents, including a guide to the selection of such reagents andmethods for their preparation are provided by Tae, H. Jr. in Meth.Enzymol. 580-609 (1983) and in the references cited therein. Theselection of the most appropriate reagent for a specific purpose fromthe wide variety of cross-linking agents available, is well within theskill of an ordinary artisan. In general, zero-length, homo- orheterobifunctional cross-linking agents are preferred for making thebispecific molecules of the present invention. Zero-length cross linkingreagents induce the direct conjugation of two binding domains withoutthe introduction of any extrinsic material. Agents that catalyze theformation of disulfide bonds belong in this category. Another example isreagents that induce the condensation of carboxy and primary aminogroups to form an amide bond, such as carbodiimides, ethylchloroformate,Woodward's reagent K1, carbonyidiimidazole, etc. Homobifunctionalreagents carry two identical functional groups, whereasheterobifunctional reagents contain two dissimilar functional groups. Avast majority of the heterobifunctional cross-linking agents contains aprimary amine-reactive group and a thiol-reactive group. A novelheterobifunctional linker for formyl to thiol coupling was disclosed byHeindel, N. D. et al., Bioconjugate Chem. 2, 427-430 (1991)]. In apreferred embodiment, the covalent cross-linking agents are selectedfrom reagents capable of forming disulfide (—S—S—), glycol(—CH[OH]—CH[OH]—), azo (—N═N—), sulfone (—S[═O₂]—), or ester (—C[═O]—O—)bridges. In a different approach, the binding domains are linked viatheir oligosaccharides. Chemical or enzymatic oxidation ofoligosaccharides on polypeptide ligands to aldehydes yields uniquefunctional groups on the molecule, which can react with compoundscontaining, for example, amines hydrazines, hydrazides, orsemicarbazides. Since the glycosylations sites are well defined inpolypeptide molecules, selective coupling via oxidized oligosaccharidemoieties will yield in a more uniform product than other couplingmethods, and is expected to have less adverse effect on the receptorbinding properties of the ligands. A carbohydrate-directedheterobifunctional cross-linking agent, 4-(4-N-maleimidophenyl)butyricacid hydrazide.HCl (MPBH), was, for example, described by Chamow et al.,J. Biol. Chem. 267, 15916-15922 (1992). MPBH can be purchased from thePierce Chemical Company, Rockford, Ill. (Product #22302), along withother reagents of similar structures, such as4-(N-maleimidomethyl)cyclohexan-1-carboxyl hydrazide.HCl (M₂C₂H; Product#22304), and 3-(2-pyridylthio)propionyl hydrazide (PDPH; Product #2230).It will be understood that the coupling of more than two bindingsequences with various linked sequences, e.g., cross-linking reagents ispossible, and is within the scope of the present invention.

In a further embodiment, in the bispecific molecules of the presentinvention the binding domains are connected by polypeptide linkersequences, and accordingly, are presented to their receptor/ligand as asingle-chain multifunctional polypeptide molecule. The polypeptidelinker functions as a “spacer” whose function is to separate thefunctional binding domains so that they can independently assume theirproper tertiary conformation. The polypeptide linker usually comprisesbetween about 5 and about 25 residues, and preferably contains at leastabout 10, more preferably at least about 15 amino acids, and is composedof amino acid residues which together provide a hydrophilic, relativelyunstructured region. Linking amino acid sequences with little or nosecondary structure work well. If desired, one or more unique cleavagesites recognizable by a specific cleavage agent (e.g. protease) may beincluded in the polypeptide linker. The specific amino acids in thespacer can vary, however, cysteines should be avoided. The spacersequence may mimic the tertiary structure of an amino acid sequencenormally linking two receptor binding domains in a native bifunctionalligand. It can also be designed to assume a desired structure, such as ahelical structure. Suitable polypeptide linkers are, for example,disclosed in WO 88/09344 (published 1 Dec. 1988), as are methods for theproduction of multifunctional proteins comprising such linkers.

In a further specific embodiment, the binding domains are connected byamphiphilic helices. It is known that recurring copies of the amino acidleucine (Leu) in gene regulatory proteins can serve as teeth that “zip”two protein molecules together to provide a dimer.

Leucine zipper was first discovered when a small segment of the proteinC/EBP was fit into a hypothetical alpha helix. Surprisingly, theleucines, which make up every seventh amino acid in this protein, linedup in a column. Subsequently, two additional, C/EBP related proteinswere identified and shown to have a similar function. One of them, GCN4is a gene regulatory protein from yeast, the other one, is the productof a proto-oncogene jun. It has been found that zipper regions associatein parallel when they combine, i.e. the leucines on apposed moleculesline up side by side. It has also been shown that non-identical proteinsmay be zippered to provide heterodimers. Such leucine zippers areparticularly suitable for preparing bispecific molecules within thescope of the invention. Alternatively, the sequence of the amphipathichelix may be taken from a four-helix bundle design, essentially asdescribed by Pack P. and Pluckthun, A., Biochemistry 31, 1579-1584(1992). For further details about molecular, e.g. leucine zippers, whichcan serve as linkers for the purpose of the present invention, see forexample: Landshculz, W. H., et al., Science 240, 1759-1764 (1988);O'Shea, E. K. et al., Science 243, 538-542 (1989); McKnight, S. L.,Scientific American 54-64, April 1991; Schmidt-Dorr. T. et al.,Biochemistry 30, 9657-9664 (1991); Blondel, A. and Bedouelle, H. ProteinEngineering 4, 457-461 (1991), Pack, P. and Pluckthun, A., supra, andthe references cited in these papers.

In a preferred embodiment, the linker comprises an immunoglobulinsequence preferably resulting in a bispecific immunoadhesin.

As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the binding specificity of a heterologousprotein (an “adhesin”) with the effector functions of immunoglobulinconstant domains. Structurally, the immunoadhesins comprise a fusion ofan amino acid sequence with the desired binding specificity which isother than the antigen recognition and binding site of an antibody (i.e.is “heterologous”), and an immunoglobulin constant domain sequence. Theadhesin part of an immunoadhesin molecule typically is a contiguousamino acid sequence comprising at least the binding site of a receptoror a ligand. The immunoglobulin constant domain sequence in theimmunoadhesins may be obtained from any immunoglobulin, such as IgG-1,IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE,IgD or IgM.

As used herein the phrase “bispecific immunoadhesin” designatesimmunoadhesins having at least two binding specificities, one of whichmay be an antigen binding site of an antibody. Bispecific immunoadhesinscan generally be assembled as hetero-multimers, and particularly ashetero-dimers,

-trimers or -tetramers, essentially as disclosed in WO 89/02922(published 6 Apr. 1989) or in EP 314,317 (published 3 May 1989). Whereasthe binding domains providing the two desired specificities in thebispecific molecules of the present invention preferably replace thevariable domains of immunoglobulins, they can also be inserted betweenimmunoglobulin heavy chain and light chain sequences such that animmunoglobulin comprising a chimeric heavy chain is obtained. In thisembodiment, the binding sequences are fused to the 3′ end of animmunoglobulin heavy chain in each arm of an immunoglobulin, eitherbetween the hinge and the CH2 domain, or between the CH2 and CH3domains. Similar constructs have been reported by Hoogenboom, H. R. etal., Mol. Immunol. 28, 1027-1037 (1991).

The term “bispecific antibody” is used herein to describe antibodymolecules which comprise two different antigen binding sites. Thebispecific antibodies may be assembled as hetero-multimers, andparticularly as hetero-dimers, -trimers or tetramers, as hereinabovedescribed for bispecific immunoadhesins.

The expression “immunoglobulin heavy chain constant domain sequencelacking a(n immunoglobulin) light chain binding site” is used todesignate an immunoglobulin heavy chain constant domain sequence fromwhich sequence elements to which the light chain is ordinarily linkedare removed or sufficiently altered (mutated) so that such binding is nolonger possible. In a preferred embodiment, the entire CH1 domain isdeleted but shorter truncations of immunoglobulin constant domains arealso suitable, provided that the section to which the light chain isordinarily disulfide-bonded or interacts with non-covalently is removed.Alternatively, the light chain binding region of an immunoglobulin heavychain constant domain may be mutated (by substitution or insertion) sothat it is no longer capable of covalent or non-covalent binding to animmunoglobulin light chain.

The terms “nucleic acid molecule encoding”, “DNA sequence encoding”, and“DNA encoding” refer to the order or sequence of deoxyribonucleotidesalong a strand of deoxyribonucleic acid. The order of thesedeoxyribonucleotides determines the order of amino acids along thepolypeptide chain. The DNA sequence thus codes for the amino acidsequence.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to a DNA encoding apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,then synthetic oligonucleotide adaptors or linkers are used in accordwith conventional practice.

The terms “replicable expression vector” and “expression vector” referto a piece of DNA, usually double-stranded, which may have inserted intoit a piece of foreign DNA. Foreign DNA is defined as heterologous DNA,which is DNA not naturally found in the host cell. The vector is used totransport the foreign or heterologous DNA into a suitable host cell.

Once in the host cell, the vector can replicate independently of thehost chromosomal DNA. and several copies of the vector and its inserted(foreign) DNA may be generated. In addition, the vector contains thenecessary elements that permit translating the foreign DNA into apolypeptide. Many molecules of the polypeptide encoded by the foreignDNA can thus be rapidly synthesized.

In the context of the present invention the expressions “cell”, “cellline”, and “cell culture” are used interchangeably, and all suchdesignations include progeny. It is also understood that all progeny maynot be precisely identical in DNA content, due to deliberate orinadvertent mutations. Mutant progeny that have the same function orbiological property as screened for in the originally transformed cellare included.

“Transformation” means introducing DNA into an organism so that the DNAis replicable, either as an extrachromosomal element or by chromosomalintegration.

“Transfection” refers to the taking up of an expression vector by a hostcell whether or not any coding sequences are in fact expressed.

The terms “transformed host cell” and “transformed” refer to theintroduction of DNA into a cell. The cell is termed a “host cell”, andit may be a prokaryotic or a eukaryotic cell. Typical prokaryotic hostcells include various strains of E. coli. Typical eukaryotic host cellsare mammalian, such as Chinese hamster ovary cells or human embryonickidney 293 cells. The introduced DNA is usually in the form of a vectorcontaining an inserted piece of DNA. The introduced DNA sequence may befrom the same species as the host cell or a different species from thehost cell, or it may be a hybrid DNA sequence, containing some foreignand some homologous DNA.

“Oligonucleotides” are short-length, single- or double-strandedpolydeoxynucleotides that are chemically synthesized by known methods(such as phosphotriester, phosphite, or phosphoramidite chemistry, usingsolid phase techniques such as those described in EP 266,032, published4 May 1988, or via deoxynucleoside H-phosphanate intermediates asdescribed by Froehler et al., Nucl. Acids Res. 14, 5399 (1986)1. Theyare then purified on polyacrylamide gels.

“Ugation” refers to the process of forming phosphodiester bonds betweentwo double stranded nucleic acid fragments (Maniatis et al., MolecularCloning: A Laboratory Manual Cold Springs Harbor Laboratory, Cold SpringHarbor, 1982, p. 146). Unless otherwise stated, ligation may beaccomplished using known buffers and conditions with 10 units of T4 DNAligase (“ligase”) per 0.5 μg of approximately equimolar amounts of theDNA fragments to be ligated. “Digestion” of DNA refers to catalyticcleavage of the DNA with an enzyme that acts only at certain locationsin the DNA. Such “restriction enzymes” recognize specific “restrictionsites”. The various restriction enzymes used herein are commerciallyavailable, and their restriction conditions, cofactors and otherrequirements as established by the enzyme suppliers were used. Ingeneral, about 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. Appropriate buffersand substrate amounts for particular restriction enzymes are specifiedby the manufacturer. Incubation times of about 1 hour at 37° C. areordinarily used, but may very in accordance with the supplier'sinstructions. After incubation, protein is removed by extraction withphenol and chloroform, and the digested nucleic acid is recovered fromthe aqueous fraction by precipitation with ethanol. Digestion with arestriction enzyme infrequently is followed with bacterial alkalinephosphatase hydrolysis of the terminal 5′ phosphates to prevent the tworestriction cleaved ends of a DNA fragments from “circularizing” orforming a closed loop that would impede insertion of another DNAfragment at the restriction site. Unless otherwise stated, digestion ofplasmids is not followed by 5′ terminal dephosphorylation. Proceduresand reagents for dephosphorylation are conventional (Maniatis et al.,Supra, pp. 133-134).

II. Availability of IFN-γ Inhibitors

A. Anti-IFN-γ and Anti-IFN-γ Receptor Antibodies

Anti-IFN-γ antibodies blocking various biological activities of nativeIFN-γ (often referred to as “neutralizing antibodies”) are known in theart, and are, for example, disclosed in the following publications:Billiau, A., Immunol. Today 9, 37-40 (1988); Hereman, H. et al., J. Exp.Med. 171, 1853-1859 (1990); Landolfo, S. et al., Science 229, 176-179(1985); Didlake, R. H. et al., Transplantation 45, 222-223 (1988),Jacob, C. O. et al., J. Exp. Med. 166, 789-803 (1987); Yong, V. W. etal., Natl. Acad. Sci. USA 88, 7016-7020 (1991)].

Antibodies to a native IFN-γ receptor which inhibit the binding ofnative IFN-γ to its receptor and thereby block IFN-γ biological activityare, for example, disclosed in EP 369,41 3 published 23 May 1990; EP393,502 published 24 Oct. 1990; EP 416,652 published 13 Mar. 1991; EP240,975 published 14 Oct. 1987; and U.S. Pat. No.4,897,264 issued 30Jan. 1990.

The following is a brief discussion of certain commonly used techniquesthat can be used for making such antibodies. Further details of theseand similar techniques are found in general textbooks, such as, forexample, Cabilly, et al., U.S. Pat. No. 4,816,567; Mage & Lamoyi, supra;Sambrook et al., Molecular Cloning: A laboratory Manual, Second Edition,Cold Spring Harbor Laboratory Press, New York, 1989; and CurrentProtocols in Molecular Biology, Ausubel et al., eds., Green PublishingAssociates and Wiley-Interscience, 1991.

Anti-IFN-γ and anti-IFN-γ receptor antibodies acting as antagonists ofIFN-γ biological action may be produced by any method known in the art.For example, the monoclonal antibodies to be used in accordance with thepresent invention may be made by the hybridoma method first described byKohler & Milstein, Nature 256:495 (1975), or may be made by recombinantDNA methods [Cabilly, et al., supra].

In the hybridoma method, a mouse or other appropriate host animal, suchas hamster is immunized with a human IFN-γ or IFN-γ receptor protein bysubcutaneous, intraperitoneal, or intramuscular routes to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the protein used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, J. MonoclonalAntibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)].

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh level expression of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOPC-21 and MPC-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2cells available from the American Type Culture Collection, Rockville,Md. USA. Human myeloma and mouse-human heteromyeloma cell lines alsohave been described for the production of human monoclonal antibodies.Kozbor, J. Immunol. 133:3001 (1984). Brodeur, et al., MonoclonalAntibody Production Techniques and Applications, pp.51-63 (MarcelDekker, Inc., New York, 1987).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against IFN-γ or IFN-γreceptor. Preferably, the binding specificity of monoclonal antibodiesproduced by hybridoma cells is determined by immunoprecipitation or byan in vitro binding assay, such as radioimmunoassay (RIA) orenzyme-linked immunoabsorbent assay (ELISA). The monoclonal antibodiesfor use in the method and compositions of the invention are those thatpreferentially immunoprecipitate IFN-γ or IFN-γ receptor that is presentin a test sample, or that preferentially bind to IFN-γ or IFN-γ receptorin a binding assay, and are capable of blocking the detrimental effectof IFN-γ in an in vitro or in vivo model of inflammatory bowel disease.

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and activity, the clones may be subclonedby limiting dilution procedures and grown by standard methods (Goding,J., Supra). Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium or RPMI-1640 medium. Inaddition, the hybridoma cells may be grown in vivo as ascites tumors inan animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography. Methods forpurification of monoclonal antibodies are well known in the art, andare, for example disclosed in Unit 11.11 of “Current Protocols inMolecular Biology”, supra, and in the references cited therein.

The amount of a specific antibody present in a hybridoma supernatant canbe quantitated by either solid-phase radioimmunoassay (RIA) or by directenzyme-linked immunoabsorbent assay (ELISA). In the solid-phaseradioimmunoassay, serially diluted antiserum is incubated in microtiterwells previously coated with IFN-γ or IFN-γ receptor. Bound antibody isdetected by employing ¹²⁵1-labeled anti-immunoglobulin antibodies. Theamount of the specific antibody in the antiserum is then determined froma standard curve generated with a specific antibody of knownconcentration. The unknown antiserum and the standard antibody areassayed in parallel. Protocols for the RIA procedure as used for isotypedetermination, and the ELISA procedure are, for example, available fromSection V of “Current Protocols in Molecular Biology”, supra, and fromthe references cited therein.

DNA encoding the monoclonal antibodies useful in the method of theinvention is readily isolated and sequenced using conventionalprocedures (e.g., by using oligonucleotide probes that are capable ofbinding specifically to genes encoding the heavy and light chains ofmurine antibodies). The hybridoma cells described hereinabove serve as apreferred source of such DNA. Once isolated, the DNA may be placed intoexpression vectors, which are then transfected into host cells such assimian COS cells, Chinese Hamster ovary (CHO) cells, or myeloma cellsthat do not otherwise produce immunoglobulin protein, to obtain thesynthesis of monoclonal antibodies in the recombinant host cells.

B. IFN-γ Variants Inhibiting the Biological Activity of Native IFN-γ

The recombinant production of IFN-γ was first reported by Gray, Goeddeland co-workers [Gray et al., Nature 295, 503-508 (1982)1, and is subjectof U.S. Pat. Nos. 4,762,791, 4,929,544, 4,727,138 and 4,925,793.Recombinant IFN-γ polypeptides lacking the first three N-terminal aminoacids (CysTyrCys), including variously truncated derivatives are, forexample, disclosed in European Publication No. 146,354. Non-human animalinterferons, including IFN-γ, are, for example, disclosed in EuropeanPublication No. 88,622. Recombinant human gamma interferon (rhIFN-γ,Actimmune®, Genentech, South San Francisco, Calif.) received FDAapproval as an immunomodulatory drug for the treatment of chronicgranulomatous disease characterized by severe, recurrent infections ofthe skin, lymph nodes, liver, lungs, and bones due to phagocytedisfunction, and is commercially available.

Amino acid sequence, glycosylation variants and covalent derivatives ofany native or recombinant IFN-γ species can be prepared by methods knownin the art. Generally, particular regions or sites of the DNA encodingIFN-γ will be targeted for mutagenesis, and thus the general methodologyemployed to accomplish this is termed site-directed mutagenesis. Themutations are made using DNA modifying enzymes such as restrictionendonucleases (which cleave DNA at particular locations), nucleases(which degrade DNA) and/or polymerases (which synthesize DNA).Restriction endonuclease digestion of DNA followed by ligation may beused to generate deletions, as described in section 15.3 of Sambrook etal., Supra.

Oligonucleotide-directed mutagenesis is the preferred method forpreparing the substitution variants of IFN-γ. It may also be used toconveniently prepare the deletion and insertion variants that can beused in accordance with this invention. This technique is well known inthe art as described by Adelman et al., (DNA. 2:183 [1983]). Theoligonucleotides are readily synthesized using techniques well known inthe art such as that described by Crea et al., (Proc. Nat'l. Acad. Sci.USA, 75:5765[1978]). The production of single-stranded templates for usein this technique is described in sections 4.21-4.41 of Sambrook et al.,Supra.

PCR mutagenesis is also suitable for making the IFN-γ variants that canbe used in the methods of the present invention. The PCR technique is,for example, disclosed in U.S. Pat. No. 4,683,195, issued 28 Jul. 1987,in section 14 of Sambrook et al., Molecular Cloning: A LaboratoryManual, second edition, Cold Spring Harbor Laboratory Press. New York1989, or in Chapter 15 of Current Protocols in Molecular Biology,Ausubel et al., eds., Greene Publishing Associates andWiley-Interscience 1991.

The DNA encoding the IFN-γ variants hereof is inserted into a replicableexpression vector for further cloning and expression. Many vectors areavailable, and selection of the appropriate vector will depend on 1)whether it is to be used for DNA amplification (cloning) or forexpression, 2) the size of the DNA to be inserted into the vector, and3) the host cell to be transformed with the vector. Each vector containsvarious components depending on its function and the host cell withwhich it is compatible. The vector components generally include, but arenot limited to, one or more of the following: a signal sequence, anorigin of replication, one or more marker genes, an enhancer element, apromoter and a transcription terminator sequence.

Suitable vectors are prepared using standard recombinant DNA procedures,isolated plasmids and DNA fragments are cleaved, tailored, and ligatedtogether in a specific order to generate the desired vectors.

After ligation, the vector with the foreign gene inserted is transformedinto a suitable host cell. The transformed cells are selected by growthon an antibiotic, commonly tetracycline (tet) or ampicillin (amp), towhich they are rendered resistant due to the presence of tet and/or ampresistance genes on the vector. If the ligation mixture has beentransformed into a eukaryotic host cell, transformed cells may beselected by the DHFR/MTX system described above. The transformed cellsare grown in culture and the plasmid DNA (plasmid refers to the vectorligated to the foreign gene of interest) is then isolated. This plasmidDNA is then analyzed by restriction mapping and/or DNA sequencing. DNAsequencing is generally performed by either the method of Messing etal., Nucleic Acids Res. 9:309 (1981) or by the method of Maxam et al.,Methods of Enzymology, 65:499 (1980).

Multicellular organisms are preferred as hosts to prepare IFN-γvariants. While both invertebrate and vertebrate cell cultures areacceptable, vertebrate cell cultures, particularly mammalian cultures,are preferable. In addition to multicellular eukaryotes, eukaryoticmicrobes such as filamentous fungi or yeast are suitable to prepareIFN-γ variants. Saccharomyces cerevisiae, or common baker's yeast, isthe most commonly used among lower eukaryotic host microorganisms.Prokaryotes are particularly useful for rapid production of largeamounts of DNA, for production of single-stranded DNA templates used forsite-directed mutagenesis, for screening many mutants simultaneously,and for DNA sequencing of the mutants generated. Suitable prokaryotichost cells include, but are not limited to, various E. coli strains.

Further details of the techniques and materials suitable for preparingthe IFN-γ amino acid sequence variants hereof are, for example,disclosed in U.S. Pat. No.5,108.901 issued 28 Apr. 1992, and in EuropeanPublication No. 146,354.

The glycosylation variants of native IFN-γ and its amino acid sequencevariants are also prepared by techniques known in the art. Glycosylationof polypeptides is typically either N-linked or O-linked. O-linkedglycoslation sites may, for example, be modified by the addition of, orsubstitution by, one or more serine or threonine residue to the aminoacid sequence of a polypeptide. For ease, changes are usually made atthe DNA level, essentially using the techniques known for thepreparation of amino acid sequence variants.

Chemical or enzymatic coupling of glycosydes to IFN-γ or its amino acidsequence variants may also be used to modify or increase the number orprofile of carbohydrate substituents. These procedures are advantageousin that they do not require production of the polypeptide that iscapable of O-linked (or N-linked) glycosylation. Depending on thecoupling mode used, the sugar(s) may be attached to (a) arginine andhistidine, (b) free carboxyl groups, (c) free hydroxyl groups such asthose of cysteine, (d) free sulfhydryl groups such as those of serine,threonine, or hydroxyproline, (e) aromatic residues such as those ofphenylalanine, tyrosine, or tryptophan or (f) the amide group ofglutamine. These methods are described in WO 87/05330 (published 11 Sep.1987), and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306(1981).

Carbohydrate moieties present on IFN-γ or an amino acid sequence variantthereof may also be removed chemically or enzymatically. Chemicaldeglycosylation requires exposure to trifluoromethanesulfonic acid or anequivalent compound. This treatment results in the cleavage of most orall sugars, except the linking sugar, while leaving the polypeptideintact. Chemical deglycosylation is described by Hakimuddin et al.,Arch. Biochem. Biophys. 259, 52 (1987) and by Edge et al., Anal.Biochem. 118, 131 (1981). Carbohydrate moieties can be removed by avariety of endo- and exoglycosidases as described by Thotakura et al.,Meth. Enzymol. 138, 350 (1987). Glycosylation is suppressed bytunicamycin as described by Duskin et al., J. Biol. Chem. 257, 3105(1982). Tunicamycin blocks the formation of protein-N-glycosydaselinkages.

Glycosylation variants can also be produced by selecting appropriatehost cells. Yeast, for example, introduce glycosylation which variessignificantly from that of mammalian systems. Similarly, mammalian cellshaving a different species (e.g. hamster, murine, insect, porcine,bovine or ovine) or tissue (e.g. lung, liver, lymphoid, mesenchymal orepidermal) origin than the source of the selectin variant, are routinelyscreened for the ability to introduce variant glycosylation.

The use of covalent derivatives of IFN-γ amino acid sequence andglycosylation variants is within the scope hereof. Such modificationsare introduced by reacting targeted amino acid residues of the IFN-γvariant with an organic derivatizing agent that is capable of reactingwith selected side chains or terminal residues, or by harnessingmechanisms of post-translational modification that function in selectedrecombinant host cells. Covalent derivatization may be instrumental inturning biologically active IFN-γ variants to derivatives which retainthe qualitative ability of the corresponding native IFN-γ to bind itsreceptor but are devoid of biological activity, or improve otherproperties, i.e. half-life, stability, etc. of the molecule. Suchmodifications are within the ordinary skill in the art and are performedwithout undue experimentation. Certain post-translational derivatizationare the result of the action of recombinant host cells on the expressedpolypeptide. Glutaminyl and asparaginyl residues are frequentlypost-translationally deamidated to the corresponding glutamyl andaspartyl residues. Alternatively, these residues are deamidated undermildly acidic conditions. Either form of these residues fall within thescope of the invention. Other post-translational modifications includehydroxylation of proline and lysine, phosphorylation of hydroxyl groupsof seryl and threonyl residues, methylation of the a-amino groups oflysine, arginine, and histidine side chains [T. E. Creighton, Proteins:Structure and Molecular Properties, W. H. Freeman Co., San Francisco pp.79-86 (1983)1, acetylation of the N-terminal amines and, in someinstances, amidation of the C-terminal carboxyl of IFN-γ. Other covalentderivatives comprise IFN-γ covalently bonded to a nonproteinaceouspolymer, such as polyethylene glycol, polypropylene glycol orpolyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192, 4,179,337, or 5,116,964.

A specific group of IFN-γ variants includes the fusion of a contiguousIFN-γ amino acid sequence antagonizing the biological action of nativeIFN-γ to a stable plasma protein, such as an immunoglobulin. Thestructure and construction of such fusion proteins (such asimmunoadhesins) will be detailed hereinbelow with reference to IFN-γreceptors. IFN-γ-stable plasma protein fusions are made in an analogousmanner.

Receptor binding domains in IFNs-γ from various species, such as humanand mouse, have been identified [see, for example, Lord, S. C. et al.,Mol. Immunol. 26,637-640 (1989); Favre, C. et al., Mol. Immunol. 26,17-25 (1989); Jarpe, M. A. and Howard, M. J., J. Immunol. 145, 3304-3309(1990); Magazine, H. I. and Johnson, H. M., Biochemistry 30, 5784-5789(1991)].

C. IFN-γ Receptor

IFN-γ receptors purified from native source or produced by techniques ofrecombinant DNA technology are known in the art, and are, for exampledisclosed in the following publications: Aguet, M. & Merlin, G., J. Exp.Med. 165, 988-999 (1987); Novick, D. et al., J. Biol. Chem. 262,8483-8487 (1987); Calderon, J. et al., Proc. Natl. Acad. Sci. USA 85,4837-4841 (1988); Basu, M. et al., Proc. Natl. Acad. Sci. USA 85,6282-6286 (1988); Aguet et al., Cell 55, 273-280 (1988); Gray, P. W. etal., Proc. Natl. Acad. Sci. USA 86, 8497-8501 (1989).

Amino acid sequence and glycosylation variants and covalentmodifications of a native (human or non-human) IFN-γ receptor can bemade following the same general techniques described hereinabove forIFN-γ.

An IFN-γ receptor amino acid sequence capable of binding native IFN-γcan also be linked to a stable plasma protein sequence as hereinbeforedefined. The stable plasma protein sequence may, for example, be animmunoglobulin sequence, preferably an immunoglobulin constant domainsequence. The resultant molecules are commonly referred to as IFN-=65receptor-immunoglobulin chimeras or, more recently, immunoadhesins.

Ordinarily, the C-terminus of a contiguous amino acid sequence of aligand-(IFN-γ-) binding domain of an IFN-γ receptor is fused to theN-terminus of a contiguous amino acid sequence of an immunoglobulinconstant region, in place of the variable region(s), however N-terminalfusions are also possible.

Typically, such fusions retain at least functionally active hinge, CH2and CH3 domains of the constant region of an immunoglobulin heavy chain.Fusions are also made to the C-terminus of the Fc portion of a constantdomain, or immediately N-terminal to the CH1 of the heavy chain or thecorresponding region of the light chain. This ordinarily is accomplishedby constructing the appropriate DNA sequence and expressing it inrecombinant cell culture. Alternatively, immunoadhesins may besynthesized according to known methods.

The precise site at which the fusion is made is not critical; particularsites are well known and may be selected in order to optimize thebiological activity, secretion or binding characteristics of theimmunoadhesins.

In a preferred embodiment, the C-terminus of a contiguous amino acidsequence which comprises the binding site(s) for IFN-γ is fused, at theN-terminal end, to the C-terminal portion of an antibody (in particularthe Fc domain), containing the effector functions of an 35immunoglobulin, e.g. immunoglobulin G₁ (IgG-1). As hereinabovementioned, it is possible to fuse the entire heavy chain constant regionto the sequence containing the binding site(s). However, morepreferably, a sequence beginning in the hinge region just upstream ofthe papain cleavage site (which defines IgG Fc chemically; residue 216,taking the first residue of heavy chain constant region to be 114 (Kobetet al., supra], or analogous sites of other immunoglobulins) is used inthe fusion. Although it was earlier thought that in immunoadhesins theimmunoglobulin light chain would be required for efficient secretion ofthe heterologous protein-heavy chain fusion proteins, it has been foundthat even the immunoadhesins containing the whole IgG 1 heavy chain areefficiently secreted in the absence of light chain. Since the lightchain is unnecessary, the immunoglobulin heavy chain constant domainsequence used in the construction of the immunoadhesins of the presentinvention may be devoid of a light chain binding site. This can beachieved by removing or sufficiently altering immunoglobulin heavy chainsequence elements to which the light chain is ordinarily linked so thatsuch binding is no longer possible. Thus, the CH1 domain can be entirelyremoved in certain embodiments of the IFN-γ receptor-immunoglobulinchimeras.

In a particularly preferred embodiment, the amino acid sequencecontaining the extracellular domain of an IFN-γ receptor is fused to thehinge region and CH2, CH3; or CH1, hinge, CH2 and CH3 domains of anIgG-1, IgG-2, IgG-3, or IgG-4 heavy chain. The construction of a typicalstructure is disclosed in Example 1.

In some embodiments, the IFN-γ receptor-immunoglobulin molecules(immunoadhesins) are assembled as monomers, dimers or multimers, andparticularly as dimers or tetramers. Generally, these assembledimmunoadhesins will have known unit structures similar to those of thecorresponding immunoglobulins. A basic four chain structural unit (adimer of two immunoglobulin heavy chain-light chain pairs) is the formin which IgG, IgA and IgE exist. A four chain unit is repeated in thehigh molecular weight immunoglobulins; IgM generally exists as apentamer of basic four-chain units held together by disulfide bonds. IgAglobulin, and occasionally IgG globulin, may also exist in a multimericform in serum. In the case of multimers, each four chain unit may be thesame or different.

It is not necessary that the entire immunoglobulin portion of the IFN-γreceptor-immunoglobulin chimeras be from the same immunoglobulin.Various portions of different immunoglobulins may be combined, andvariants and derivatives of native immunoglobulins can be made ashereinabove described with respect to IFN-γ, in order to optimize theproperties of the immunoadhesin molecules. For example, immunoadhesinconstructs in which the hinge of IgG-1 was replaced with that of IgG-3were found to be functional and showed pharmacokinetics comparable tothose of immunoadhesins comprising the entire IgG-1 heavy chain.

The IFN-γ receptor-immunoglobulin immunoadhesins may contain twodifferent binding domains not ordinarily present in an immunoglobulinmolecule. In a specific embodiment, the two binding domains are twodifferent IFN-γ binding amino acid sequences from an IFN-γ receptor. Inanother embodiment, only one binding domain is from an IFN-γ receptor,whereas the other is a different polypeptide sequence not ordinarilypresent in an immunoglobulin molecule. The second binding domainpreferably is an amino acid sequence capable of binding a polypeptideimplicated in the initiation or development of IBD. This second bindingsequence preferably is from a second IFN-γ inhibitor, an IL-1 inhibitor,a TNF-α inhibitor, a CD11a/18 inhibitor, a CD11b/18 inhibitor, or anL-selectin inhibitor, and may comprise the antibody-antigen combiningsite of an antibody to IFN-γ, IL-1, TNF-α, CD11a/1 8, CD11b/18,L-selectin, or to the respective receptors/ligands, or may, for example,be an IL-1 receptor or type 1 or type 2 TNF-α receptor (TNF-R1 orTNF-R2) amino acid sequence. If both binding domains in a chimericimmunoglobulin molecule are from antibodies of different bindingspecificities, the structure is commonly referred to as bispecificantibody.

Diagrams illustrating the possible structures of mono- and bispecificimmunoadhesins are, for example, disclosed in U.S. Pat. No. 5,116,964issued 26 May 1992, as are chains or basic units of varying structurewhich may be utilized to assemble the monomers and hetero- andhomo-multimers of immunoglobulins in such constructs.

Bispecific immunoadhesins and antibodies are also disclosed in EP355,068 published 21 Feb. 1990, and PCT application publication no. WO91/05871 published 2 May 1991.

In certain cases it may be advantageous to prepare trimeric bispecificimmunoadhesins, where in one arm of a Y-shaped immunoglobulin, the firstheavy chain constant domain (CH1) is removed or altered to eliminate itsability to covalently bind to an immunoglobulin light chain (i.e. thelight chain binding site is eliminated or inactivated), while in theother arm the light chain binding site within the CH1 domain isretained, and is covalently linked to an immunoglobulin light chain. Ineach arm, the heavy chain constant domain sequences are fused to“binding domains” (replacing the heavy chain variable domains) which aredifferent from one another. One of the binding domains is from an IFN-γantagonist, whereas the other binding domain may be selected from thecytokine antagonists referred to hereinabove. The resultant structure isa trimer with two different binding specificities (heterotrimer)composed of an immunoglobulin heavy chain constant domain sequence fusedto a first binding domain and a second immunoglobulin heavy chainconstant domain sequence fused to a different binding domain andcovalently linked to an immunoglobulin light chain.

Two or more of the heterotrimers may be covalently linked to each otherto provide a multimeric structure. Generally, these assembled bispecificimmunoadhesins will have known unit structures. A three-chain unit maybe repeated similarly to the higher molecular weight immunoglobulins.

In another embodiment, fusion proteins with two specificities may bemade by fusing the IFN-γ receptor amino acid sequence to the 3′ end ofan antibody heavy chain, where the antibody may, for example, be ananti-IFN-γ antibody, or any of the other antibodies referred tohereinabove. The IFN-γ receptor amino acid sequence may, for example, beinserted wither between the hinge and CH2 domains, or between the CH2and CH3 domains of the immunoglobulin heavy chain. The chimericimmunoglobulin heavy chain-IFN-γ receptor gene may then be introducedinto an immunoglobulin light chain secreting transfectoma cell line,producing the ligrit chain of the desired antibody. The construction ofsimilar structures was reported by Hoogenboom. H. R., Mol. Immunol. 28,1027-1037 (1991).

Linear fusion proteins with dual specificity, comprising the fusion ofan IFN-γ receptor amino acid sequence to an antibody sequence may beprepared essentially as described by Traunecker et al., EMBO 10,3655-3659 (1991).

Traunecker et al., designed a single-chain polypeptide, containing theFvCD3, the two N-terminal domains of CD4 and C-kappa, designated Janusin(CD4-FvCD3-Ckappa). This bispecific linear molecule was purified byusing anti-kappa affinity columns. Linear molecules comprising an IFN-γreceptor amino acid sequence can be made in an analogous manner.

Immunoglobulins and certain variants thereof are known and many havebeen prepared in recombinant cell culture. For example, see U.S. Pat.No. 4,745,055; EP 256,654; Faulkner et al., Nature 298:286 (1982); EP120,694; EP 125,023; Morrison, J. Immun. 123:793 (1979); Köhler et al.,Proc. Nat'l. Acad. Sci. USA 77:2197 (1980); Raso et al., Cancer Res.41:2073 (1981); Morrison et al., Ann. Rev. Immunol. 2:239 (1984);Morrison, Science 229:1202 (1985); Morrison et al., Proc. Nat'l. Acad.Sci. USA 81:6851 (1984); EP 255,694; EP 266,663; and WO 88/03559.Reassorted immunoglobulin chains also are known (see for example U.S.Pat. No. 4,444,878; WO 88/03565; and EP 68,763 and references citedtherein), as are synthetic antibody binding sites (Fv analogues)produced by protein engineering (see e.g. Huston, J. S. et al., Proc.NatI. Acad. Sci. USA 85, 5879-5883 (1988), and U.S. Pat. No. 5,091,513issued 25 Feb. 19921.

The selection of the immunoglobulin that provides the (heavy chain)constant domain sequences in the IFN-γ receptor immunoglobulinimmunoadhesins of the present invention, is a matter of choice, largelydepending on the motivation for constructing them. If the extension ofplasma half-life of the IFN-γ receptor is a consideration,immunoglobulins of IgG-1, IgG-2 and IgG-4 isotypes are good candidates,as they all have in vivo half-lives of 21 days, as opposed to IgG-3which has an in vivo half-life of 7 days. Further differences that mightbe advantages or detriments in certain situations are, for example, incomplement activation. IgG-1, IgG-2 and IgG-3 all activate complement,however, IgG-2 is significantly weaker at complement activation thanIgG-1 and does not bind to Fc receptors on mononuclear cells orneutrophils, while IgG-3 shows better complement activation than IgG-1.IgG-1 has only four serologically-defined allotypic sites, two of which(G1 m1 and 2) are in the Fc portion; for two of these sites (G1m1 and17) one allotype is non-immunogenic. In contrast, there are 12serologically defined allotypes in IgG-3, all of which are in the Fcportion, only three of which (G3m5, 11 and 21) have an allotype which isnon-immunogenic. Thus, for repeated and long-term therapeuticapplications, immunoadhesins with IgG-1 derived constant domainsequences are preferred. It is possible to use immunoglobulins fromdifferent classes or isotypes in the two arms of the bispecificimmunoadhesin molecule. It is further possible to combine sequences fromvarious immunoglobulin classes or isotypes in the same arm of abispecific molecule, or in both arms of a mono- or bispecificimmunoadhesin.

The IFN-γ receptor-immunoglobulin chimeras may be prepared by standardtechniques of recombinant DNA technology detailed hereinabove. Anotheralternative is to chemically synthesize the gene encoding the chimerasusing one of the methods described in Engels et al., Agnew. Chem. Int.Ed. Enl. 28, 716 (1989). These methods include triester, phosphite,phosphoramidite and H-phosphonate methods, PCR and other autoprimermethods, and oligonucleotide syntheses on solid supports.

Fusion proteins comprising other stable plasma proteins can be made bymethods known in the art. For example, fusion proteins comprisingN-terminal fragments of human serum albumin (HSA) as stable plasmaprotein are disclosed in EP 399,666 published 28 Nov. 1990.

D. Selection of IFN-γ Inhibitors

The IFN-γ inhibitor useful in performing the method of the presentinvention may be tested by a number of in vitro and in vivo methods.

Binding Assays

The ability of an inhibitor to bind IFN-γ (such as in the case of IFN-γreceptor or anti-IFN-γ antibodies) can, for example, be tested byequilibrium binding analysis, essentially as described by Ashkenazi, A.et al., Proc. Natl. Acad. Sci. USA 88, 10535-10539 (1991). According tothis method, the IFN-γ inhibitor candidate is immobilized ontomicrotiter wells coated with goat anti-human IgG Fc antibody, and isincubated with detectably labeled IFN-γ. A similar method is describedin Examples 5B and 5C of EP 393,502 published 24 Oct. 1990.Alternatively or in addition, the affinity of binding can be tested incompetition binding assays, such as, for example, disclosed byFountoulakis, M. et al., J. Biol. Chem. 265, 13268-13275 (1990). Theability of an IFN-γ inhibitor as defined for the purpose of the presentinvention, to inhibit IFN-γ binding to its receptor (e.g. IFN-γ variantsand anti-IFN-γ receptor antibodies) can be tested in an analogousmanner. If the label is radioactive, the binding can be quantitated by aγ or β scintillation counter.

IFN-γ Biological Activity Assays

The ability of IFN-γ inhibitors to block the biological activity ofIFN-γ can be tested in any conventional in vitro assays of IFN-γ action.For example, the ability of an inhibitor to block the induction ofexpression of specific antigens by IFN-γ can be assayed, essentially asdescribed in Example 2. In another group of assays (antiviral assays),the ability of an inhibitor candidate to block the protective effect ofIFN-γ against viral infection is tested. A specific antiviral assay isdisclosed in Example 2. Alternatively or in addition, one can use a hostimmune-response model to test the ability of an IFN-γ inhibitor to blockendogenous IFN-γ, for example as disclosed in Example 2 hereinafter. Thesame example also discloses a suitable mouse model for testing IFN-γinhibitor action.

In Vitro Model of IBD

An in vitro model of IBD has been developed by MacDonald, T. andco-workers, and in described by Braegger, C. P. and MacDonald, T. inChapter 8 of “Immunology of Gastrointestinal Disease”, supra, and hasearlier been reported by MacDonald, T. and Spencer, J., J. Exp. Med.167, 1341-1349 (1988). In this model, small explants (1-2 mm across) ofhuman fetal gut tissue (small or large bowel) containing T lymphocytesat the stage of 15-20 weeks gestation are cultured. The human fetal gutcan be maintained in organ culture for several weeks with retention ofmorphology, epithelial cell renewal and enterocyte function. All of theT cells in the explant can be activated by culturing in the presence ofpokeweed mitogen or monoclonal anti-CD3 antibodies. The gross appearanceof the explants shows major changes as a result of T cell activation.The changes in the small bowel explants as a result of T cell activationare reminiscent of the mucosal change seen in early stages of Crohn'sdisease, and the goblet cell depletion seen in colon explants is also afeature of ulcerative colitis. This model can be used to study theinteraction of T cells with the gut epithelium and specifically, toobserve responses to T cell activation, such as secretion of IFN-γ, andblocking of IFN-γ secretion by giving an IFN-γ inhibitor candidate priorto or at the time of T cell activation.

Animal Models of IBD

The first group of animal models of IBD includes animals spontaneouslydeveloping diseases reminiscent of some forms of IBD. Spontaneous animalmodels include C3H/HeJ mouse, Japanese waltzing mice, swine dysenteryand equine colitis, caused by C. difficile, and the cotton top tamarin.The diseases that these animals suffer have recently been subdividedinto five types, two of which resemble UC. Of these models, tamarin arepreferred, as a large proportion of these animals have some form of gutdisorder, and many of them also develop bowel cancer, as do patientswith UC.

In another approach, various irritants, such as ethanol, acetic acid,formalin, immune complexes, trinitrobenzene sulphonic acid (TNBS),bacterial products or carrageenan are used to generate acute or chronicinflammation. A model of this kind has been developed by Wallace, J. andcoworkers [Morris et al., Gastroenterology 96, 795 (1989)].

According to a third approach, transgenic animals are used to model IBD.Most human patients who have ankylosing spondylitis also carry the genefor HLA-B27. It has been observed that such patients are at greater riskof developing IBD. HLA-B27 transgenic rats, which were attempted tomodel spondyloarthropathies, in addition to the joint disease, alsoshowed symptoms of chronic inflammation of the bowel which, though notidentical, had many similarities with CD. Accordingly, the HL-B27transgenic rats can be used to model IBD.

Another suitable transgenic animal model is based on IL-10 “knockout”mice. IL-10 is produced by TH2 cells, stimulates B cells to produceantibody, downregulates macrophages reducing the production of IL-1,IL-6, IL-8 and TNF-α, and shifts the balance of antigen presentationfrom macrophages to B cells. IL-10 also reduces the production of IFN-γ,hence reducing the activity of TH1 cells and natural killer cells. Micetreated from birth with anti-IL-10 antibody (given i.p. 3-times weekly)show no changes in body weight or histology of major tissues. The numberand proportions of B and T cell lymphocytes are also normal. There is,however, a dramatic reduction in IgA production, whereas theconcentrations of IgG-2a and IgG-2b are increased. In addition, analmost total depletion of peritoneal B cells, which are a special B cellpopulation carrying the marker Ly-1, was observed. These B cells arecontinuously derived from bone marrow, have a limited immunoglobulinrepertoire which is not subject to somatic mutation, and are responsiblefor much of the IgM found in plasma. The depletion of these specific Bcells may be due to the increased level of IFN-γ that are produced inthe anti-IL-10 antibody-treated mice. This is supported by theobservation that if IFN-γ is given at the same time as the anti-IL-10antibody, the Ly-1 B cells survive.

Usually the screening for antagonists suitable for the purpose of thepresent invention includes a combination of the foregoing assays, theresults obtained in vitro and in vivo models of IBD being the mostconclusive.

E. Pharmaceutical Compositions

The IFN-γ inhibitors of the present invention, including the bispecificmolecules herein, are usually administered as pharmaceuticalcompositions, usually formulated in dosage forms by methods known in theart; for example, see Remington's Pharmaceutical Sciences, MackPublishing Company, Easton, Pa., 15th Edition 1975. For parenteraladministration, the antagonists are typically formulated in the form ofinjectable solutions, suspensions or emulsions, in admixture with asuitable pharmaceutically acceptable vehicle and optionally otherpharmaceutically acceptable additives. Typical vehicles include saline,dextrose solution, Ringer's solution, etc., but non-aqueous vehicles mayalso be used.

Pharmaceutical compositions comprising IFN-γ receptor amino acidsequences and antagonist anti-IFN-γ receptor antibodies along withsuitable dosages and dose rates are, for example, disclosed in EP369,413 published 23 May 1990; EP 393,502 published 24 Oct. 1990; EP416,652 published 13 Mar. 1991; EP 240,975 published 14 Oct. 1987; and

U.S. Pat. No. 4,897,264 issued 30 Jan. 1990.

The formulation of IFN-γ variants is preferably liquid, and isordinarily a physiological salt solution or dextrose solution, togetherwith conventional stabilizers and/or excipients. IFN-γ compositions mayalso be provided as lyophilized powders. IFN-γ-containing pharmaceuticalcompositions are, for example, disclosed in U.S. Pat. No. 4,727,138issued 23 Feb. 1988, U.S. Pat. No. 4,762,791 issued 9 Aug. 1988, U.S.Pat. No. 4,925,793 issued 15 May 1990, U.S. Pat. No. 4,929,553 issued 29May 1990, U.S. Pat. No. 4,855,238 issued 8 Aug. 1989. A typicalformulation may contain IFN-γ (20×10⁶ U) at 1.0 or 0.2 mg/ml, 0.27 mg/mlsuccinic acid, and disodium succinate hexahydrate 0.73 ml/injection atpH 5.0. Preferred IFN-γ formulations and dose ranges are disclosed inU.S. Pat. No. 5,151,265 issued 29 Sep. 1992. The formulations anddosages for anti-IFN-γ receptor antibodies are similar, and can bedetermined without undue experimentation.

The actual dose for each particular IFN-γ inhibitor will depend on themedical condition to be treated, the pathological condition and clinicaltolerance of the patient involved, the properties of the preparationsemployed, including their activity and biological half-life, etc. Itwill be appreciated that the practitioner will adjust the dose in linewith clinical experience.

The inhibitors of the present invention many be applied prophylacticallyto patients known to be at risk of developing a disease to be treated orsubsequent to the onset of the disease. Such patients are, for example,those diagnosed with and possibly treated for inflammatory bowel diseaseon at least one earlier occasion but asymptomatic at the time ofadministration.

The IFN-γ inhibitors of the present invention may be administered incombination with other therapeutics potentially useful in the treatmentof a condition characterized by a decrease in the IgA/IgG ratio, such asinflammatory bowel disease. These other therapeutics may beantiinflammatory agents, such as sulfasalazine, corticosteroids,6-mercaptopurine/azathioprine; immunosuppressants such as cyclosporine,prostaglandin inhibitors; superoxide dismutase; IL-1 receptorantagonists; anti-ELAM-1 (E-selectin) antibodies; inhibitors of VCAM-1binding to eosinophils; anti-CD18 antibodies. The administration may besimultaneous or consecutive, and includes the administration offormulations comprising one or more of these and similar therapeutics incombination with one or more IFN-γ inhibitor. Alternatively, suchfurther therapeutics may be included in the fusion polypeptides(bispecific immunoadhesins, antibodies, linear molecules) of the presentinvention.

Further details of the invention are set forth in the followingnon-limiting examples.

EXAMPLE 1

Construction of Human and Murine IFN-γ Receptor-Immunoadhesins

IFN-γ receptor (IFNγR) immunoadhesins were constructed from plasmidspRK-H-γR or pRK-M-γR [Gibbs, V. C. et al., Mol. Cell. Biol. 11,5860-5866. (1991)], encoding the human and murine IFNγR, respectively,and plasmid pRKCD4₂Fc₁, encoding CD4-IgG-1 (Byrn, R. A. et al., Nature344, 667-670 (1990)]. Briefly, this CD4-IgG-1 construct consists ofresidues 1-180 of the mature human CD4 protein fused to human IgG-1sequences beginning at aspartic acid 216 (taking amino acid 114 as thefirst residue of the heavy chain constant region [Kabat et al., supra])which is the first residue of the IgG-1 hinge after the cysteine residueinvolved in heavy-light chain bonding, and ending with residue 441. Thismolecule contains the CD4 V1 and V2 domains, linked to the hinge and Fc(CH2 and CH3) domains of human IgG-1, and is designated CD4₂Fc1.

Complementary DNA (cDNA) fragments encoding the human or murine IFNγR(each with its natural sign sequence) were generated by digestion of therespective plasmids with CIa I and Hind III. Plasmid pRKCD4₂Fc₁ wasdigested with Cla I and Nhe I to remove most of the CD4 sequence whileretaining the human IgG-1 heavy chain hinge region and CH2 and CH3domain sequences. Intermediate plasmids were constructed by insertingeach IFNγR sequence 5′ of the IgG-1 sequence and in the same readingorientation, by ligating the respective Cla I sites and by blunting andligating the Hind III and Nhe I sites. Next, the remaining CD4 sequenceand the sequence encoding the transmembrane and intracellular portion ofeach IFNγR were removed by oligonucleotide-directed deletion mutagenesisto create the exact junction between the extracellular portion of eachIFNγR and the IgG-1 hinge.

At the junctions are the codons for glycine-231 or aspartic acid-216 ofthe human or murine IFNγR and the additional 226 codons of human IgG-1heavy chain. Thus, the mature hIFNγR-IgG-1 and mIFNγR-IgG-1 polypeptidesare 448 and 44 amino acids long, respectively. Deletion mutagenesis wasdone using synthetic oligonucleotides complementary to the 18 bases oneach side of the desired junctions as primers, and the aboveintermediate plasmids as templates, as described by Ashkenazi, A. etal., Proc. Natl. Acad. Sci. USA 88, 10535-10539 (1991). The finalstructure was confirmed by DNA sequencing.

The IFNγR-IgG-1 immunoadhesins (also referred to as IFNγR-IgG) wereexpressed in human embryonic kidney (HEK) 293 cells by transienttransfection using a modification of the calcium phosphate precipitationmethod [Marsters, S. A. et al., J. Biol. Chem. 267, 5747-5750 (1992)].The immunoadhesins were purified from serum-free cell culturesupernatants in a single step by affinity chromatography onStapylococcus aureus Protein A taking advantage of the binding of theIgG-1 Fc domain to Protein A. Bound proteins were eluted with 50 mMsodium citrate pH 3/20% (w/v) glycerol and the eluted pools wereneutralized with 0.05 volumes of 3M Tris HCl pH 8 to 9. IFNγR-IgG-1processed in this way was more than 95 % pure. The obtained constructscomprise the fusion of the extracellular portion of human (h) or murine(m) IFNγR with the hinge region and CH2 and CH3 domains of IgG-1 heavychain (FIG. 1A)

We examined the subunit structure of the immunoadhesins bypolyacrylamide gel electrophoresis (FIG. 1B-D). Under non-reducingconditions, a predominant band with a relative molecular mass (Mr) of160-220 kDa was observed for both human and murine IFNγR-IgG-1; underreducing conditions, this band was absent and a new band of 65-90 kDaappeared (FIG. 1B). This indicates that each IFNγR-IgG-1 is secreted asa disulfide-bonded homodimer. The broad range of Mr probably is due tosubstantial glycosylation of the immunoadhesins, as there are fivepotential N-linked glycosylation sites in the IFNγR region [Aguet, M. etal., Cell 55, 273-280 (1988); Cofano, F. et al., J. Biol. Chem. 265,4064-4071 (1990); Gray, R. W. et al., Proc. Natl. Acad. Sci. USA 86,8497-8501 (1989); Hemmi, S. et al., Proc. Natl. Acad. Sci. USA 86,9901-9905 (1989); Kumar, C. S. et al., J. Biol. Chem. 264, 17939-17946(1989); Munro, S. and Maniatis, T., Proc. NatI. Acad. Sci. USA 86,9248-9252 (1989)] and one in the IgG-1 region [Byrn, R. A., 1990,supra]. Western blots using polyclonal anti-IgGγFc antibodies confirmedthe identity of 160-220 kDa band as IFNγR-IgG-1; immunoreactivity withthe anti-IgG antibodies was diminished upon reduction, probably due todisruption of the disulfide-stabilized domain structure of the Fcportion (FIG. 1C). Ligand blots with [¹²⁵I]-labeled human or murineIFN-γ revealed the same Mr band, which was not labeled in the presenceof excess cold IFN-γ from the respective species (FIG. 1D). Theseresults demonstrate the presence of an IgG-1 heavy chain and of afunctional receptor domain that binds IFN-γ specifically in bothIFNγR-IgG-1 molecules. The human and murine IFN-γ receptors are about50% identical at the amino acid level, and both bind IFN-γ in aspecies-specific manner [Finbloom, D. S. et al., J. Immunol. 135,300-305 (1985); Ucer, U. et al., Int. J. Cancer. 36, 103-108 (1991)].

EXAMPLE 2

Testing of IFN-γ Inhibitor Activity

1. Assays

IFNγ binding assays. The binding of IFNγR-IgGs to IFN-γ was analyzedessentially as described [Ashkenazi et al., Proc. Natl. Acad. Sci. USA88: 10535-10539 (1991)]. Each IFNγR-IgG (1 μg/ml) was immobilized ontomicrotiter wells coated with goat anti-human IgG Fc antibody. Human ormurine IFNγR-IgG was incubated with recombinant,[¹²⁵I]-labeled human (h)or murine (m) IFN-γ (radioiodinated using lactoperoxidase to a specificactivity of 20-30 μCi/μg) in phosphate buffered saline (PBS) containing1% bovine serum albumin (BSA) for 1 h at 24° C. Nonspecific binding wasdetermined by omitting IFNγR-IgGs.

IFN-γ induction of gene expression assays. The biological activity ofIFNγR-IgG in vitro was assessed by testing its ability to block theinduction of expression of specific antigens by IFNγ. Human HeLa cellsor mouse L929 cells were grown to 30% confluency and incubated for 48 hrat 37° C./5% CO₂ with 10 ng/ml of hIFN-γ or mIFN-γ, respectively.IFNγR-IgG from the respective species, or CD4-IgG as a negative control,also was added to the cultures. The expression of ICAM-1 by the humancells and of the class I MHC antigen H-2K^(k) by the murine cells wasthen analyzed by flow cytometry, using specific fluoresceinatedantibodies. Propidium iodide was added immediately before analysis togate out nonviable cells.

IFNγ antiviral activity assays. The ability of IFNγR-IgG to block theprotective effect of IFN-γ against infection of cells withencephalomyocarditis virus (EMCV) also was tested as a measure of theimmunoadhesin's inhibitory activity in vitro. We used a modification ofa method described previously [Rubinstein et al, J. Virol. 37: 755-758(1981)]. Human A549 or murine L929 cells were plated in microtiterdishes (2×10⁴ cells/well) and incubated at 37° C./5% CO₂ for 24 hr.Then, recombinant human or murine IFN-γ was added at concentrations of0.125 or 0.5 ng/ml, respectively, concomitant with human or murineIFNγR-IgG, and the incubation was continued for 24 h. EMCV was thenadded to the cells at 1 multiplicity of infection unit per well. Thesurvival of the cells was quantitated by crystal violet exclusion 18-24hr later.

Murine listeriosis. The ability of IFNγR-IgG to block endogenous IFN-γin vivo was investigated in a host immune-response model, using Listeriamonocytogenes infection in mice [Buchmier and Schreiber, Proc. Natl.Acad. Sci USA 82: 7404-7408 (1985); Farber and Peterkin, MicrobiologicalReviews 55: 476-511 (1991)]. Female C57BL/6×DBA/2F. (BDF1) mice, statedto be free of infection by adventitious viral agents, were obtained at 6weeks of age from Charles River (Portage, Mich.). The L. monocytogeneswas maintained as described previously [Haak-Frendscho, et al., Infect.Immun. 57: 3014-3021 (1989)]. Log-phase bacteria were suspended intryptone phosphate broth containing 20% glycerol and stored as aliquotsat −70° C. Mice were given an intraperitoneal (i.p.) injection ofvehicle, CD4-IgG as a negative control, or mIFNγR-IgG. Immediatelyafter, each mouse received 4×10⁴ freshly thawed bacteria in 0.2 mlpyrogen-free PBS by intravenous (i.v.) injection via a lateral tailvein. Three days later, the mice were euthanized and their spleens andlivers were removed and homogenized in separate sterile tissue grinderscontaining 1 ml sterile water. The homogenates were diluted serially andplated onto agar. Plates were incubated 24 hr at 37° C., whereafter thebacterial colonies were enumerated. Mouse model for contact-sensitivity.The ability of IFNγR-IgG to block endogenous IFN-γ in vivo wasinvestigated further using a mouse model for contact-sensitivity. Balb/cmice (Charles River, Portage, Mich.) were anesthetized by i.p. injectionof Ketamine/Xylazene. A 3×3 cm square patch on the abdomen was shaved,where 200 μg of 2,4-dinitro-1-fluorobenzene (DNFB) was applied topically(sensitization). Five days later, 20 μg of DNFB was applied topically toboth sides of the left pinna, whereas diluent was applied to both sidesof the right pinna (challenge). Ten hours later, the mice were injectedi.v. via a lateral tail vein with 2 μCi of [¹²⁵I]UdR. Sixteen hourslater, the mice were euthanized, the pinnae were removed at thehairline, and [¹²⁵I] radioactivity was counted as a measure oflymphocyte proliferation and migration to the pinnae. Mice treated withIgG, immunoadhesins, or-monoclonal antibodies were injected i.v. withvehicle or agent 30 min prior to sensitization and 30 min prior tochallenge.

2. Results

Binding of IFNγR-IgG to IFN-γ. We investigated the binding of the humanand murine immunoadhesins to IFN-γ by equilibrium binding analysis (FIG.2). Each IFNγR-IgG exhibited specific and saturable binding to[¹²⁵I]-labeled IFN-γ from the autologous species. Scatchard analysisindicated dissociation constant (K_(d)) values of 1.5 nM for hIFNγR-IgGwith [¹²⁵I]hIFN-γ and 3.3 nM for mIFNγR-IgG with [¹²⁵I]mIFN-γ.Competition binding assays (data not shown) showed no significantinhibition by mIFN-γ of hIFN-γ binding to hIFNγR-IgG, or by hIFN-γ ofmIFN-γ binding to mIFNγR-IgG. These results indicate that eachimmunoadhesin binds IFN-γ in a species-specific manner, consistent withprevious reports [Finbloom et al., J. Immunol. 135: 300-305 (1985);Ucer, et al., Int. J. Cancer 36: 103-108 (1985)]. The IFN-γ bindingaffinity of IFNγR-IgG is comparable to that reported for recombinantsoluble IFNγR (Fountoulakis, et al., J. Biol. Chem. 265: 13268-13275(1990)]. Therefore, the attachment of the IFNγR extracellular domain tothe IgG heavy-chain does not hinder IFN-γ binding.

IFNγR-IgG blocks IFNγ in vitro. We tested the ability of human andmurine IFNγR-IgG to inhibit immunomodulatory and antiviral effects ofIFN-γ on cultured cells (FIG. 3). The induction of ICAM-1 by hIFN-γ inhuman HeLa cells could be blocked completely by hIFNγR-IgG, withhalf-maximal inhibition (IC₅₀) at about 13 nM of immunoadhesin. Incontrast, CD4-IgG, used as a negative control, had no effect (FIG. 3A).Similarly, the induction of class I MHC antigen H-2K^(k) by mIFN-γ inmouse L929 cells could be blocked completely by mIFNγR-IgG, with an IC₅₀of about 10 nM, whereas CD4-IgG had no effect (FIG. 3B). The cytopathiceffect of encephalomyocarditis virus (EMCV) can be prevented by additionof IFN-γ. In EMCV-infected human A549 cells, hIFNγR-IgG blocked theantiviral effect of hIFN-γ completely, with an IC₅₀ of about 8 nM (FIG.3C). Similarly, mIFNγR-IgG blocked the antiviral effect of mIFN-γ inEMCV-infected mouse L929 cells, with an lC₅₀ of about 10 nM (FIG. 3D).In all experiments performed, IFN═R-IgG from the alternate species hadno significant effect on antiviral activity, confirming the speciesselectivity of the interaction between the immunoadhesins and IFN-γ.These results demonstrate the ability of IFNγR-IgG to block efficientlyand completely the interaction of IFN-γ with its cell-surface receptor,thus preventing the cytokine from exerting biological effects on cellsin vitro.

IFNγR-IgG blocks IFN-γ in vivo. To assess the ability of IFNγR-IgG toneutralize endogenous IFN-γ in vivo, we used a mouse model for the hostimmune response against infection with Listeria monocytogenes; in whichIFN-γ is known to play a central role (Buchmier et al., supra; Farber etal., supra). Mice were given an i.p. injection of vehicle, CD4-IgG, ormIFNγR-IgG (200 μg), followed immediately with an LD₅₀ i.v. challenge ofL. monocytogenes. Three days later, the number of bacterial colonyforming units (CFU) in the spleens and livers of the mice weredetermined. Whereas CD4-IgG did not affect the number of CFU in eitherorgan significantly, treatment with IFNγR-IgG resulted in about aten-fold increase in the number of CFU in both spleen and liver (FIG.4). Sequential analysis of bacterial loads over a period of 7 daysfollowing infection (data not shown) indicated that IFNγR-IgG affectedthe extent, and not the kinetics, of infection. These results indicatethat the action of endogenous IFN-γ against infection with L.monocytogenes was inhibited effectively by IFNγR-IgG, thus demonstratingthe ability of this immunoadhesin to block IFN-γ biological activity invivo.

A relevant model for testing the ability of IFNγR-IgG to inhibit adverseconsequences of inappropriate IFN-γ production in vivo iscontact-sensitivity in mice. This immune response is based upon theinteraction of an antigen with primed T-cells, and represents tissuedamage resulting from inappropriate cell-mediated immunity, in whichcytokines such as IFN-γ, TNF-α and IL-1 play an important role[Askenase, P. W. (1988) Effector and Regulatory Mechanisms inDelayed-Type Reed, E. F. Ellis and N. F. Atkinson, eds. C. V. Mosby, St.Louis; Enk and Katz, Proc. Natl. Acad. Sci. USA 89: 1398-1402 (1992);Belisto et al., J. Immunol. 143: 1530-1536 (1989); Piguet et al., J.Exp. Med. 173: 673-679 (1991)1. We sensitized mice to the hapten DNTP bytopical administration to the abdomen. Five days later, the mice werechallenged with DNFP by topical application to one pinna, whereasdiluent was applied to the other pinna. Ten hours later, the micereceived an i.v. injection of [¹²⁵I]UdR. Sixteen hours after isotopeinjection, the mice were euthanized and the pinnae were removed andanalyzed for [¹²⁵I] incorporation, as a measure of lymphocyteproliferation and migration to the site of contact with antigen.Injection of mIFNγR-IgG (200 μg i.v. at priming and at challenge)resulted in about 44% inhibition of the response (Table 1). TABLE 1Inhibition of IFNγ by IFNγR-IgG in a mouse model for contact-sensitivityDose* Response† Inhibition‡ Treatment (μg/mouse) (Fold) (%) Experiment 1Vehicle — 1.21 ± 0.08 — IgG 200  4.35 ± 0.54 0 IFNγR-IgG 200  2.96 ±0.46 44.3 Experiment 2 Vehicle — 0.91 ± 0.15 — IgG 50 4.42 ± 0.52 0IFNγR-IgG 50 3.51 ± 0.37 25.9 TNFR-IgG 50 3.54 ± 0.28 25.1 IFNγR-IgG +TNFR-IgG 50 + 50 2.80 ± 0.25 46.2 Anti-mIFNγ 50 3.79 ± 0.49 17.9Anti-TNF 50 3.77 ± 0.41 18.5 Anti-IFNγ + Anti-TNF 50 + 50 2.37 ± 0.2758.4*Given i.v. at priming and at challenge.†The numbers represent the ratio between [¹²⁵I] radioactivity measuredin the antigen-painted pinna and the vehicle-painted pinna. Values aremeans ± SEM (n = 6 mice per treatment group).‡Relative to the IgG control.At a lower dose of 50 μg, less inhibition was observed (26%). It islikely that the inhibition of the response by IFNγR-IgG was not completebecause other cytokines are involved in elicitating contact-sensitivityalong with IFN-γ. Indeed, treatment with a TNF receptor immunoadhesin[TNFR1-IgG; Ashkenazi, A. et al., Proc. Natl. Acad. Sci. USA 88,10535-10539 (1991)] at a dose of 50 μg gave inhibition similar toIFNγR-IgG at 50 μg; moreover, a combination of the two immunoadhesins(50 μg each) resulted in an approximately additive inhibition of theresponse (Table 1). For comparison, we tested the effect of anti-IFN-γand anti-TNF-α monoclonal antibodies. The extent of inhibition by theseantibodies, when given individually or in combination was comparable tothat observed with the IFNγR and TNFR immunoadhesins (Table 1). Theseresults indicate that the contribution of IFN-γ to thecontact-sensitivity response is inhibited effectively by IFNγR-IgG, thusdemonstrating further the ability of this immunoadhesin to block IFN-γbiological activity in vivo.

In conclusion, each IFNγR-IgG immunoadhesin bound IFN-γ in aspecies-specific manner with nanomolar affinity, comparable torecombinant soluble IFN-γ receptors. In cultured cells, IFNγR-IgG wasable to block completely IFN-γ-induced expression of ICAM1 and MHC classI antigen, and IFN-γ antiviral activity. In mice, IFNγR-IgG inhibitedthe function of IFN-γ produced as a major cytokine in response tobacterial infection and in the elicitation of contact sensitivity. Thus,IFNγR-IgG is a specific and effective inhibitor of IFN-Y both in vitroand in vivo.

EXAMPLE 3

Inhibition of Gut Damage

The ability of IFN-γ receptor-IgG-1 chimeras to prevent gut damagecharacteristic of inflammatory bowel disease was tested in a model offetal intestine explants. In this model, human fetal intestine explantsare stimulated with staphylococcal exterotoxin B (SEB). This activatesthe lamina propria T cells expressing Vβ3 and the local cell-mediatedimmune response causes gut damage, which is seen as an increase inepithelial proliferation and the loss of matrix glycosaminoglycans dueto the release of proteases and endoglycosydases by activatedmacrophages. This latter phenomenon is believed to be an important stepin the production of ulcers in the gut.

Human small intestine was obtained within two hours of surgicaltermination from the Medical Research Council Foetal Tissue Bank. Thisstudy was conducted under conditions approved by the Hackney andDistrict Health Authority ethical committee.

The small intestine (ileum) of a 15.3 week old fetus was dissected into2-mm² explants and these were then cultured (20 per dish in 7 mls) withstaphylococcal enterotoxin B (SEB) for 4 days at 37° C. in a 95% oxygen,5% CO₂ atmosphere in 7 ml of serum-free CMRL-1066 medium (FlowLaboratories Inc., McLean, Va.), modified according to Autrup et al.,[Rosenzweig and Kanwar, Lab. Invest. 47, 177-184 (1982)1, but with theomission of hydrocortisone.

Separate cultures were set up with and anti-IL-2 antibodies (100 μl ofcell culture supernatant, corresponding to only about 100-200 ng/ml ofantibody), and a human IFN-γ receptor-IgG-1 chimera prepared asdescribed in Example 1. These were added at the same time as SEB. At theend of the four days cultivation period, the tissues were snap frozen inliquid nitrogen, and stored at −70° C.

Frozen sections were cut to 6 μm sections, and the presence anddistribution of lamina propria glycosaminoglycans (GAGs) visualized bysilver staining [Klein et al., Histochem. J. 25, 291-298 (1993); Kleinet al., J. Cell. Sci. 102, 81-832 (1992)]. Specifically, anionic siteswere visualized with a 5 nm gold-conjugated poly-L-lysine probe (BiocellResearch Laboratories, Cardiff, U.K.) diluted 1 in 100 inphosphate-buffered saline, pH 1-2, free of calcium and magnesium. Theprobe was applied to the sections for 60 minutes, washed off withdeionized water, and developed with a silver enhancer (Biocell) forabout 15 minutes at room temperature. The slides were counterstained inMayer's haemalum and mounted in Apathy's medium.

To quantify GAGs, multiple fields were scanned on an optical integrateddensitometer. Units were chosen arbitrarily. The higher the number, thegreater the staining and the greater the amount of GAGs in the tissue.

In the explants treated with SEB, the activated T cells secretedlymphokines which activated lamina propria acrophages. These thensecrete metalloproteinases which degrade the extracellular matrix.

The data set forth in FIG. 5 show that the IFN-γ receptor-IgG-1 chimerawas effective in preventing gut damage in this system. Although the testalso included a minor amount of anti-IL-2 antibodies, the effect wasclearly attributable to the IFN-γ-IgG-1 chimera, as in separateexperiments, anti-IL-2 antibodies were found ineffective in this system.

Although the foregoing refers to particular preferred embodiments, itwill be understood that the present invention is not so limited. It willoccur to those ordinarily skilled in the art that various modificationsmay be made to the disclosed embodiments without diverting from theoverall concept of the invention. All such modifications are intended tobe within the scope of the present invention.

1-29. (canceled)
 30. An isolated bispecific molecule comprising a firstbinding domain connected to a second binding domain, wherein the firstbinding domain comprises an IFN-γ inhibitor comprising an anti-IFN-γantibody, and the second binding domain comprising an IFN-γ inhibitorselected from the group consisting of an IFN-γ receptor, anextracellular domain of an IFN-γ receptor capable of binding IFN-γ, anIFN-γ receptor variant sequence capable of binding IFN-γ, an anti-IFN-γreceptor antibody, and anti-IFN-γ antibody sequence.
 31. The isolatedbispecific molecule or claim 30, wherein the second binding domaincomprises an IFN-γ receptor sequence.
 32. The isolated bispecificmolecule of claim 31, wherein the second binding domain comprises anextracellular domain of an IFN-γ receptor sequence capable of bindingIFN-γ.
 33. The isolated bispecific molecule of claim 30, wherein thesecond binding domain comprises an anti-IFN-γ antibody sequence.
 34. Theisolated bispecific molecule of claim 30, wherein the second bindingdomain comprises an anti-IFN-γ receptor antibody sequence.
 35. Theisolated bispecific molecule of claim 30, wherein the second bindingdomain comprises an IFN-γ receptor variant sequence.
 36. The isolatedbispecific molecule of claim 30, wherein said first and second bindingdomains are connected with a linker.
 37. The isolated bispecificmolecule of claim 36, wherein said linker comprises a polypeptide. 38.The isolated bispecific molecule of claim 37, wherein said polypeptidecomprises an immunoglobulin sequence.
 39. The isolated bispecificmolecule of claim 38, wherein said bispecific molecule is a bispecificimmunoadhesin.
 40. The isolated bispecific molecule of claim 39, whereinsaid first binding domain is fused to a first immunoglobulin constantdomain sequence, and said second binding domain is fused to a secondimmunoglobulin constant domain sequence.
 41. The isolated bispecificmolecule of claim 40, wherein said first binding domain is fused at itsC-terminus to the N-terminus of a first immunoglobulin heavy chainconstant domain sequence comprising at least a hinge region and theC_(H)2 and C_(H)3 domains of an IgG-1, IgG2 or IgG-3 immunoglobulin. 42.The isolated bispecific molecule of claim 41, wherein said secondbinding domain is fused at its C-terminus to the N-terminus of a secondimmunoglobulin heavy chain constant domain sequence comprising at leasta hinge region and the C_(H)2 and C_(H)3 domains of an IgG-1, IgG-2 orIgG-3 immunoglobulin.
 43. The isolated bispecific molecule of claim 42,wherein the fusion comprising said first binding domain and said firstimmunoglobulin heavy chain constant domain sequence is disulfide-linkedto the fusion comprising said second binding domain and said secondimmunoglobulin heavy chain constant domain sequence.
 44. An isolatednucleic acid encoding a bispecific molecule according to claim
 30. 45. Areplicable expression vector comprising the nucleic acid of claim 44.46. A host cell comprising a replicable expression vector of claim 45.47. A process comprising culturing a host cell of claim
 46. 48. Acomposition comprising a bispecific molecule according to claim 30 inadmixture with a carrier.
 49. A method for treatment of ulcerativecolitis and Crohn's disease in a patient comprising administering to thepatient an effective amount of a bispecific molecule of claim
 30. 50. Anisolated bispecific molecule comprising a first binding domain connectedto a second binding domain, wherein the first binding domain comprisesan IFN-γ inhibitor and the second binding domain comprising an IL-1inhibitor, a TNF-α inhibitor, a CD11a/18 inhibitor, a L-selectininhibitor, or a VLA-4 inhibitor.
 51. An isolated bispecific molecule ofclaim 50, wherein the second binding domain is an IL-1 inhibitorselected from the group consisting of an IL-1 receptor, an extracellulardomain of an IL-1 receptor capable of binding IL-1, an IL-1 receptorvariant sequence capable of binding IL-1, an anti-IL-1 receptorantibody, and an anti-IL-1 antibody.
 52. An isolated bispecific moleculeof claim 50, wherein the second binding domain is a TNF-α inhibitorselected from the group consisting of an anti-TNF-α antibody, ananti-TNF-α receptor antibody, a type 1 or a type 2 TNF-α receptor, and aTNF-α receptor immunoadhesin.
 53. An isolated bispecific molecule ofclaim 50, wherein the second binding domain is a CD11a/18 inhibitorselected from the group consisting of a CD11a/18 receptor, anextracellular domain of a CD11a/18 receptor capable of binding CD11a/18,a CDl la/18 receptor variant sequence capable of binding CD11a/18, ananti-CD11 a/18 receptor antibody, and anti-CD11a/18 antibody.
 54. Anisolated bispecific molecule of claim 50, wherein the second bindingdomain is a L-selectin inhibitor selected from the group consisting of aL-selectin receptor, an extracellular domain of a L-selectin receptorcapable of binding L-selectin, a L-selectin receptor variant sequencecapable of binding L-selectin, an anti-L-selectin receptor antibody, andanti-L-selectin antibody.
 55. An isolated bispecific molecule of claim50, wherein the second binding domain is a VLA-4 inhibitor selected fromthe group consisting of a VLA-4 receptor, an extracellular domain of aVLA-4 receptor capable of binding VLA-4, a VLA-4 receptor variantsequence capable of binding VLA-4, an anti-VLA-4 receptor antibody, andanti-VLA-4 antibody.
 56. The isolated bispecific molecule of claim 50,wherein said first and second binding domains are connected with alinker.
 57. The isolated bispecific molecule of claim 56, wherein saidlinker comprises a polypeptide.
 58. The isolated bispecific molecule ofclaim 50, wherein said polypeptide comprises an immunoglobulin sequence.59. The isolated bispecific molecule of claim 58, wherein saidbispecific molecule is a bispecific immunoadhesin.
 60. An isolatednucleic acid encoding a bispecific molecule according to claim
 50. 61. Areplicable expression vector comprising the nucleic acid of claim 60.62. A host cell comprising a replicable expression vector of claim 61.63. A process comprising culturing a host cell of claim
 62. 64. Acomposition comprising a bispecific molecule according to claim 50 inadmixture with a carrier.
 65. A method for treatment of ulcerativecolitis and Crohn's disease in a patient comprising administering to thepatient an effective amount of a bispecific molecule of claim
 50. 66. Amethod for treatment of ulcerative colitis and Crohn's disease in apatient comprising administering to the patient an effective amount ofan interferon-gamma (IFN-γ) inhibitor selected from the group consistingof an IFN-γ receptor, an anti-IFN-γ antibody, an anti-IFN-γ receptorantibody, and an IFN-γ variant, wherein said IFN-γ variant is a variantof human IFN-γ that retains the receptor binding domain andwhich-inhibits the binding of native IFN-γ to its native receptor.
 67. Amethod for treating ulcerative colitis and Crohn's disease in a patientcomprising administering to the patient an effective amount of (a)interferon-gamma (IFN-γ), an anti-IFN-γ receptor antibody, or an IFN-γvariant, wherein said IFN-γ variant is a variant of human IFN-γ thatretains the receptor binding domain and inhibits the binding of nativeIFN-γ to its native receptor, and (b) IL-1 inhibitor, a TNF-α inhibitor,a CD11a/18 inhibitor, a L-selectin inhibitor, or a VLA-4 inhibitor. 68.The method of claim 67, wherein (b) is a TNF-α inhibitor selected fromthe group consisting of an anti-TNF-α antibody, an anti-TNF-α receptorantibody, a type 1 or a type 2 TNF-α receptor, and a TNF-α receptorimmunoadhesin.
 69. The method of claim 67, wherein (b) is an IL-1inhibitor selected from the group consisting of an IL-1 receptor, anextracellular domain of an IL-1 receptor capable of binding IL-1, anIL-1 receptor variant sequence capable of binding IL-1, an anti-IL-1receptor antibody, and an anti-IL-1 antibody.
 70. The method of claim67, wherein (b) is a CD11a/18 inhibitor selected from the groupconsisting of a CD11a/18 receptor, an extracellular domain of a CD11a/18receptor capable of binding CD11a/18, a CD11a/18 receptor variantsequence capable of binding CD11a/18, an anti-CD11a/18 receptorantibody, and anti-CD11a/18 antibody.
 71. The method of claim 67,wherein (b) is a L-selectin inhibitor selected from the group consistingof a L-selectin receptor, an extracellular domain of a L-selectinreceptor capable of binding L-selectin, a L-selectin receptor variantsequence capable of binding L-selectin, an anti-L-selectin receptorantibody, and anti-L-selectin antibody.
 72. A The method of claim 67,wherein (b) is a VLA-4 inhibitor selected from the group consisting of aVLA-4 receptor, an extracellular domain of a VLA-4 receptor capable ofbinding VLA-4, a VLA-4 receptor variant sequence capable of bindingVLA-4, an anti-VLA-4 receptor antibody, and anti-VLA-4 antibody.
 73. Themethod of claim 66, wherein the anti-IFN-γ antibody is a humanizedantibody.
 74. The method of claim 67, wherein the anti-IFN-γ antibody isa humanized antibody.